Sdf-1 delivery for treating ischemic tissue

ABSTRACT

Provided herein are methods of treating a cardiomyopathy in a subject by administering directly to, or expressing locally in, a weakened, ischemic, and/or peri-infarct region of myocardial tissue of the subject an amount of SDF-1 effective to cause functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test, or New York Heart Association (NYHA) functional classification. Also provided are methods of treating critical limb ischemia in a subject by administering a DNA plasmid encoding human SDF-1 by direct injection into the affected limb.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/556,595, filed Jul. 24, 2012, which is a continuation of U.S.application Ser. No. 13/393,141, filed Jun. 7, 2012 as entry into theU.S. national stage of International Application No. PCT/US2010/047175,filed Aug. 30, 2010, which, in turn, claims priority from U.S.Provisional Application Nos. 61/237,775, filed Aug. 28, 2009, and61/334,216, filed May 13, 2010. The subject matter of the foregoingapplications is incorporated herein by reference in its entirety for allpurposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 22, 2013 andis named JUVE0076.txt and is 5,952 bytes in size.

FIELD OF THE INVENTION

This application relates to SDF-1 delivery methods and compositions fortreating a cardiomyopathy and to the use of SDF-1 delivery methods andcompositions for treating an ischemic cardiomyopathy. This applicationadditionally relates to SDF-1 delivery methods and compositions fortreating critical limb ischemia.

BACKGROUND OF THE INVENTION

Ischemia is a condition wherein the blood flow is completely obstructedor considerably reduced in localized parts of the body, resulting inanoxia, reduced supply of substrates and accumulation of metabolites.Although the extent of ischemia depends on the acuteness of vascularobstruction, its duration, tissue sensitivity to it, and developmentalextent of collateral vessels, dysfunction usually occurs in ischemicorgans or tissues, and prolonged ischemia results in atrophy,denaturation, apoptosis, and necrosis of affected tissues.

In ischemic cardiomyopathies, which are diseases that affect thecoronary artery and cause myocardial ischemia, the extent of ischemicmyocardial cell injury proceeds from reversible cell damage toirreversible cell damage with increasing time of the coronary arteryobstruction.

Critical limb ischemia (CLI) represents the most advanced stage ofatherosclerotic, lower extremity peripheral vascular disease (PVD) andis associated with high rates of cardiovascular morbidity, mortality,and major amputation. The incidence of CLI is estimated to be 125,000 to250,000 patients per year in the United States and is expected to growas the population ages. PVD prevalence increases dramatically with ageand affects approximately 20% of Americans age 65 and older. The currentstandard of care for individuals with CLI includes lower extremityrevascularization, either through open peripheral surgical procedures,endovascular techniques, or lower extremity amputation (i.e., ifrevascularization has failed or is not feasible). The 1-year mortalityrate of patients with CLI is 25% and may be as high as 45% in those whohave undergone amputation. Despite advanced techniques in vascular andsurgical procedures, a considerable proportion of patients with CLI arenot suitable for revascularization. Of these patients, only 20 to 30percent of CLI patients are undergoing treatment, 30% will require majoramputation and 23% will die within 3 months.

Gene therapy strategies to open blocked vessels or stimulateangiogenesis are under active investigation. In a trial of 6 patientswith CLI who were scheduled to undergo major amputation, patients wereassigned to receive NV1FGF, a non-viral gene therapy expressingfibroblast growth factor 1 (FGF1), 3 to 5 days prior to amputation. NVwas administered at 8 intramuscular sites with doses of 0.4 to 4 mg.This trial documented FGF1 transgene expression at all doses up to 3 cmfrom the injection sites, and that disseminated plasmid into bloodvessel was rapidly degraded. In the phase II double-blind,placebo-controlled, multicenter trial conducted in the USA(TALISMAN₂O₂), 71 patients with CLI were assigned to receive placebo or1 of 5 treatment regimens of 2 to 16 mg of NV delivered via 8intramuscular injections in the affected leg. This trial showed that upto 16 mg of intramuscular NV was safe and well tolerated. The primaryendpoint of TcPO2 was increased over baseline in both NV 1 FGF- andplacebo-treated patients, but there was improvement in ulcer healing inthe NV 1 FGF-treated patients. Similarly, Nikol et al. demonstrated in adouble-blind, randomized, placebo controlled study of 125 patients thatNV1FGF significantly reduced (two-fold) the risk of all amputations,major limb amputations, and there was a trend towards decreased risk ofdeath. These clinical trials demonstrate a safety window of nakedplasmid DNA therapies similar to those in our proposed clinical study.

SUMMARY OF THE INVENTION

This application relates to a method of treating a cardiomyopathy in asubject. The cardiomyopathy can include, for example, cardiomyopathiesassociated with a pulmonary embolus, a venous thrombosis, a myocardialinfarction, a transient ischemic attack, a peripheral vascular disorder,atherosclerosis, and/or other myocardial injury or vascular disease. Themethod includes administering directly to or expressing locally in aweakened, ischemic, and/or peri-infarct region of myocardial tissue ofthe subject an amount of SDF-1 effective to cause functional improvementin at least one of the following parameters: left ventricular volume,left ventricular area, left ventricular dimension, cardiac function,6-minute walk test (6MWT), or New York Heart Association (NYHA)functional classification.

In an aspect of the application, the amount of SDF-1 administered to theweakened, ischemic, and/or peri-infarct region is effective to causefunctional improvement in at least one of left ventricular end systolicvolume, left ventricular ejection fraction, wall motion score index,left ventricular end diastolic length, left ventricular end systoliclength, left ventricular end diastolic area, left ventricular endsystolic area, left ventricular end diastolic volume, 6-minute walk test(6MWT), or New York Heart Association (NYHA) functional classification.In another aspect of the application, the amount of SDF-1 administeredto the weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular end systolic volume. In a further aspect of theapplication, the amount of SDF-1 administered to the weakened, ischemic,and/or peri-infarct region is effective to improve left ventricularejection fraction.

In some aspects of the application, the amount of SDF-1 administered tothe weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular end systolic volume by at least about 10%. Inother aspects of the application, the amount of SDF-1 administered tothe weakened, ischemic, and/or peri-infarct region is effective toimprove left ventricular end systolic volume by at least about 15%. Instill further aspects of the application, the amount of SDF-1administered to the weakened, ischemic, and/or peri-infarct region iseffective to improve left ventricular end systolic volume by at leastabout 10%, improve left ventricular ejection fraction by at least about10%, improve wall motion score index by at least about 5%, improve sixminute walk distance at least about 30 meters, and improve NYHA class byat least 1 class. In a further aspect of the application, the amount ofSDF-1 administered to the weakened, ischemic, and/or peri-infarct regionis effective to improve left ventricular ejection fraction by at leastabout 10%.

In another aspect of the application, the amount of SDF-1 administeredto the weakened, ischemic, and/or peri-infarct region is effective tosubstantially improve vasculogenesis of the weakened, ischemic, and/orperi-infarct region by at least about 20% based on vessel density ormeasured by myocardial perfusion imaging (e.g., SPECT or PET) with animprovement in summed rest score, summed stress score, and/or summeddifference score of at least about 10%. The SDF-1 can be administered byinjecting a solution comprising SDF-1 expressing plasmid in theweakened, ischemic, and/or peri-infarct region and expressing SDF-1 fromthe weakened, ischemic, and/or peri-infarct region. The SDF-1 can beexpressed from the weakened, ischemic, and/or peri-infarct region at anamount effective to improve left ventricular end systolic volume.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of the solution with each injection comprising about 0.33mg/ml to about 5 mg/ml of SDF-1 plasmid solution. In one example, theSDF-1 plasmid can be administered to the weakened, ischemic, and/orperi-infarct region in at least about 10 injections. Each injectionadministered to the weakened, ischemic, and/or peri-infarct region canhave a volume of at least about 0.2 ml. The SDF-1 can be expressed inthe weakened, ischemic, and/or peri-infarct region for greater thanabout three days.

In an example application, each injection of solution comprising SDF-1expressing plasmid can have an injection volume of at least about 0.2 mland an SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml. In another aspect of the application, at least onefunctional parameter of the of the heart can be improved by injectingthe SDF-1 plasmid into the weakened, ischemic, and/or pen-infarct regionof the heart at an injection volume per site of at least about 0.2 ml,in at least about 10 injection sites, and at an SDF-1 plasmidconcentration per injection of about 0.33 mg/ml to about 5 mg/ml.

In a further example, the amount of SDF-1 plasmid administered to theweakened, ischemic, and/or peri-infarct region that can improve at leastone functional parameter of the heart is greater than about 4 mg. Thevolume of solution of SDF-1 plasmid administered to the weakened,ischemic, and/or peri-infarct region that can improve at least onefunctional parameter of the heart is at least about 10 ml.

In another aspect of the application, the subject to which the SDF-1 isadministered can be a large mammal, such as a human or pig. The SDF-1plasmid can be administered to the subject by catheterization, such asintra-coronary catheterization or endoventricular catheterization. Themyocardial tissue of the subject can be imaged to define the area ofweakened, ischemic, and/or pen-infarct region prior to administration ofthe SDF-1 plasmid, and the SDF-1 plasmid can be administered to theweakened, ischemic, and/or peri-infarct region defined by the imaging.The imaging can include at least one of echocardiography, magneticresonance imaging, coronary angiogram, electroanatomical mapping, orfluoroscopy.

The application also relates to a method of treating a myocardialinfarction in a large mammal by administering SDF-1 plasmid to thepen-infarct region of the myocardium of the mammal by catheterization,such as intra-coronary catheterization or endo-ventricularcatheterization. The SDF-1 administered by catheterization can beexpressed from the peri-infarct region at an amount effective to causefunctional improvement in at least one of the following parameters: leftventricular volume, left ventricular area, left ventricular dimension,cardiac function, 6-minute walk test (6MWT), or New York HeartAssociation (NYHA) functional classification.

In an aspect of the application, the amount of SDF-1 administered to theperi-infarct region is effective to cause functional improvement in atleast one of left ventricular end systolic volume, left ventricularejection fraction, wall motion score index, left ventricular enddiastolic length, left ventricular end systolic length, left ventricularend diastolic area, left ventricular end systolic area, left ventricularend diastolic volume, 6-minute walk test (6MWT), or New York HeartAssociation (NYHA) functional classification.

In another aspect of the application, the amount of SDF-1 administeredto the peri-infarct region is effective to improve left ventricular endsystolic volume. In a further aspect of the application, the amount ofSDF-1 administered to the weakened, ischemic, and/or peri-infarct regionis effective to improve left ventricular ejection fraction.

In some aspects of the application, the amount of SDF-1 administered tothe peri-infarct region is effective to improve left ventricular endsystolic volume by at least about 10%. In other aspects of theapplication, the amount of SDF-1 administered to the peri-infarct regionis effective to improve left ventricular end systolic volume by at leastabout 15%. In still further aspects of the application, the amount ofSDF-1 administered to the peri-infarct region is effective to improveleft ventricular end systolic volume by at least about 10%, improve leftventricular ejection fraction by at least about 10%, improve wall motionscore index by about 5%, improve six minute walk distance at least about30 meters, or improve NYHA class by at least 1 class. In a furtheraspect of the application, the amount of SDF-1 administered to theweakened, ischemic, and/or peri-infarct region is effective to improveleft ventricular ejection fraction by at least about 10%.

In another aspect of the application, the amount of SDF-1 administeredto the peri-infarct region is effective to substantially improvevasculogenesis of the peri-infarct region by at least about 20% based onvessel density.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of the solution with each injection comprising about 0.33mg/ml to about 5 mg/ml of SDF-1 plasmid/solution. In one example, theSDF-1 plasmid can be administered to the weakened, ischemic, and/orperi-infarct region in at least about 10 injections. Each injectionadministered to the weakened, ischemic, and/or peri-infarct region canhave a volume of at least about 0.2 ml. The SDF-1 can be expressed inthe weakened, ischemic, and/or peri-infarct region for greater thanabout three days.

In an example application, each injection of solution comprising SDF-1expressing plasmid can have an injection volume of at least about 0.2 mland an SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml. In another aspect of the application, at least onefunctional parameter of the of the heart can be improved by injectingthe SDF-1 plasmid into the weakened, ischemic, and/or peri-infarctregion of the heart at an injection volume per site of at least about0.2 ml, in at least about 10 injection sites, and at an SDF-1 plasmidconcentration per injection of about 0.33 mg/ml to about 5 mg/ml.

In a further example, the amount of SDF-1 plasmid administered to theweakened, ischemic, and/or peri-infarct region that can improve at leastone functional parameter of the heart is greater than about 4 mg. Thevolume of solution of SDF-1 plasmid administered to the weakened,ischemic, and/or peri-infarct region that can improve at least onefunctional parameter of the heart is at least about 10 ml.

The application further relates to a method of improving leftventricular end systolic volume in a large mammal after myocardialinfarction. The method includes administering SDF-1 plasmid to theperi-infarct region of the mammal by endo-ventricular catheterization.The SDF-1 can be expressed from the peri-infarct region at an amounteffective to cause functional improvement in left ventricular endsystolic volume.

In some aspects of the application, the amount of SDF-1 administered tothe peri-infarct region is effective to improve left ventricular endsystolic volume by at least about 10%. In other aspects of theapplication, the amount of SDF-1 administered to the peri-infarct regionis effective to improve left ventricular end systolic volume by at leastabout 15%. In still further aspects of the application, the amount ofSDF-1 administered to the peri-infarct region is effective to improveleft ventricular end systolic volume by at least about 10%, improve leftventricular ejection fraction by at least about 10%, improve wall motionscore index by about 5%, improve six minute walk distance at least about30 meters, or improve NYHA class by at least 1 class.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of the solution with each injection comprising about 0.33mg/ml to about 5 mg/ml of SDF-1 plasmid/solution. In one example, theSDF-1 plasmid can be administered to the weakened, ischemic, and/orperi-infarct region in at least about 10 injections. Each injectionadministered to the weakened, ischemic, and/or peri-infarct region canhave a volume of at least about 0.2 ml. The SDF-1 can be expressed inthe weakened, ischemic, and/or peri-infarct region for greater thanabout three days.

In an example application, each injection of solution comprising SDF-1expressing plasmid can have an injection volume of at least about 0.2 mland an SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml. In another aspect of the application, left ventricularend systolic volume of the of the heart can be improved can be improvedat about 10% by injecting the SDF-1 plasmid into the weakened, ischemic,and/or peri-infarct region of the heart at an injection volume per siteof at least about 0.2 ml, in at least about 10 injection sites, and atan SDF-1 plasmid concentration per injection of about 0.33 mg/ml toabout 5 mg/ml.

In a further example, the amount of SDF-1 plasmid administered to theweakened, ischemic, and/or peri-infarct region that can improve leftventricular end systolic volume is greater than about 4 mg. The volumeof solution of SDF-1 plasmid administered to the weakened, ischemic,and/or peri-infarct region that can improve left ventricular endsystolic volume of the heart is at least about 10 ml.

This application additionally relates to a method of treating criticallimb ischemia in a subject. The method includes administering ACRX-100(also known as JVS-100), the sterile biological product (composed ofplasmid ACL-01110Sk, the naked DNA plasmid encoding human SDF-1 cDNA,and 5% dextrose) by direct injection into the ischemic limb. Preferably,the injections are made directly into the muscle tissue, for example,into the upper leg (quadriceps muscles) and/or lower leg (primarilygastrocnemius muscle) using multiple injection sites.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the application will become apparentto those skilled in the art to which the application relates uponreading the following description with reference to the accompanyingdrawings.

FIG. 1 is a chart illustrating luciferase expression for varying amountsand volume of DNA in a porcine model;

FIG. 2 is a chart illustrating % change of left ventricular end systolicvolume for various amounts of SDF-1 plasmid using a porcine model ofcongestive heart failure 30 days following SDF-1 injection;

FIG. 3 is a chart illustrating % change of left ventricular ejectionfraction for various amounts of SDF-1 plasmid using a porcine model ofcongestive heart failure 30 days following SDF-1 injection;

FIG. 4 is a chart illustrating % change in wall motion score index forvarious amounts of SDF-1 plasmid using a porcine model of congestiveheart failure 30 days following SDF-1 injection;

FIG. 5 is a chart illustrating % change of left ventricular end systolicvolume for various amounts of SDF-1 plasmid using a porcine model ofcongestive heart failure 90 days following SDF-1 injection; and

FIG. 6 is a chart illustrating % change of vessel density for variousamounts of SDF-1 plasmid using a porcine model of congestive heartfailure 30 days following SDF-1 injection.

FIG. 7 is a schematic diagram of an SDF-1 plasmid vector.

FIG. 8 is an image showing plasmid expression over a substantial portionof a porcine heart.

FIG. 9 is a chart illustrating left ventricular end systolic volume atbaseline and 30 days post-initial injection. All groups show similarincreases in left ventricular end systolic volume at 30 days. N=3 forall data points. Data presented as mean±SEM.

FIG. 10 is a chart illustrating left ventricular ejection fraction atbaseline and 30 days post-initial injection. All groups show lack ofimprovement in left ventricular ejection fraction. N=3 for all datapoints. Data presented as mean±SEM.

FIG. 11 is an image of luciferase expression in ischemic rat leg 3 daypost-injection (A) and a chart of time course of ACRX-100 vectorexpression in a rodent HLI model (B).

FIG. 12 is an image of the bioluminescence of rabbit hindlimb muscle 3days post-injection with ACL-01110L luciferase plasmid DNA.

FIG. 13 is a chart of ACL-01110L dosing parameters in rabbit hindlimb.

FIG. 14 is an example of angiograms and scoring of ischemic hindlimb ofrabbit at baseline (A and C) and 30 days post-injection with ACRX-100 (Band D).

FIG. 15 is a chart of the percent change in angiographic score 30 and 60days post-injection with ACRX-100, normalized to control per group.

FIG. 16 is a chart of ACRX-100 biodistribution post-cardiac injection.

FIG. 17 is a chart of the relationship between SDF-1 and CXCR4expression after ischemic injury. CXCR4 is the primary receptor forSDF-1.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the application(s) belong. All patents, patentapplications, published applications and publications, Genbanksequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. In the event that there are a pluralityof definitions for terms herein, those in this section prevail. Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thisapplication belongs. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, SpringerVerlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Methodsfor chemical synthesis of nucleic acids are discussed, for example, inBeaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucciet al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleicacids can be performed, for example, on commercial automatedoligonucleotide synthesizers. Immunological methods (e.g., preparationof antigen-specific antibodies, immunoprecipitation, and immunoblotting)are described, e.g. in Current Protocols in Immunology, ed. Coligan etal., John Wiley & Sons, New York, 1991; and Methods of ImmunologicalAnalysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.Conventional methods of gene transfer and gene therapy can also beadapted for use in the application. See, e.g., Gene Therapy: Principlesand Applications, ed. T. Blackenstein, Springer Verlag, 1999; GeneTherapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins,Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. C. P.Hodgson, Springer Verlag, 1996.

Where reference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, ACRX-100 is the sterile biological product composed ofACL-01110Sk, the naked DNA plasmid encoding human SDF-1 cDNA, and 5%dextrose. (ACRX-100 may also be referred to as JVS-100 in theapplication).

As used herein, “nucleic acid” refers to a polynucleotide containing atleast two covalently linked nucleotide or nucleotide analog subunits. Anucleic acid can be a deoxyribonucleic acid (DNA), a ribonucleic acid(RNA), or an analog of DNA or RNA. Nucleotide analogs are commerciallyavailable and methods of preparing polynucleotides containing suchnucleotide analogs are known (Lin et al. (1994) Nucl. Acids Res.22:5220-5234; Jellinek et al. (1995) Biochemistry 34: 11363-11372;Pagratis et al. (1997) Nature Biotechnol. 15:68-73). The nucleic acidcan be single-stranded, double-stranded, or a mixture thereof. Forpurposes herein, unless specified otherwise, the nucleic acid isdoublestranded, or it is apparent from the context.

As used herein, “DNA” is meant to include all types and sizes of DNAmolecules including cDNA, plasmids and DNA including modifiednucleotides and nucleotide analogs.

As used herein, “nucleotides” include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides, such as,but are not limited to, phosphorothioate nucleotides and deazapurinenucleotides and other nucleotide analogs.

As used herein, the term “subject” or “patient” refers to animals intowhich the large DNA molecules can be introduced. Included are higherorganisms, such as mammals and birds, including humans, primates,rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs,cats, dogs, horses, chicken and others.

As used herein “large mammal” refers to mammals having a typical adultweight of at least 10 kg. Such large mammals can include, for example,humans, primates, dogs, pigs, cattle and is meant to exclude smallermammals, such as mice, rats, guinea pigs, and other rodents.

As used herein, “administering to a subject” is a procedure by which oneor more delivery agents and/or large nucleic acid molecules, together orseparately, are introduced into or applied onto a subject such thattarget cells which are present in the subject are eventually contactedwith the agent and/or the large nucleic acid molecules.

As used herein, “delivery,” which is used interchangeably with“transduction,” refers to the process by which exogenous nucleic acidmolecules are transferred into a cell such that they are located insidethe cell. Delivery of nucleic acids is a distinct process fromexpression of nucleic acids.

As used herein, a “multiple cloning site (MCS)” is a nucleic acid regionin a plasmid that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. “Restriction enzyme digestion” refers to catalyticcleavage of a nucleic acid molecule with an enzyme that functions onlyat specific locations in a nucleic acid molecule. Many of theserestriction enzymes are commercially available. Use of such enzymes iswidely understood by those of skill in the art. Frequently, a vector islinearized or fragmented using a restriction enzyme that cuts within theMCS to enable exogenous sequences to be ligated to the vector.

As used herein, “origin of replication” (often termed “ori”), is aspecific nucleic acid sequence at which replication is initiated.Alternatively, an autonomously replicating sequence (ARS) can beemployed if the host cell is yeast.

As used herein, “selectable or screenable markers” confer anidentifiable change to a cell permitting easy identification of cellscontaining an expression vector. Generally, a selectable marker is onethat confers a property that allows for selection. A positive selectablemarker is one in which the presence of the marker allows for itsselection, while a negative selectable marker is one in which itspresence prevents its selection. An example of a positive selectablemarker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscalorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al., Virology52:456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual(1989); Davis et al., Basic Methods in Molecular Biology (1986); Chu etal., Gene 13:197 (1981). Such techniques can be used to introduce one ormore exogenous DNA moieties, such as a nucleotide integration vector andother nucleic acid molecules, into suitable host cells. The termcaptures chemical, electrical, and viral-mediated transfectionprocedures.

As used herein, “expression” refers to the process by which nucleic acidis translated into peptides or is transcribed into RNA, which, forexample, can be translated into peptides, polypeptides or proteins. Ifthe nucleic acid is derived from genomic DNA, expression may, if anappropriate eukaryotic host cell or organism is selected, includesplicing of the mRNA. For heterologous nucleic acid to be expressed in ahost cell, it must initially be delivered into the cell and then, oncein the cell, ultimately reside in the nucleus.

As used herein, “genetic therapy” involves the transfer of heterologousDNA to cells of a mammal, particularly a human, with a disorder orconditions for which therapy or diagnosis is sought. The DNA isintroduced into the selected target cells in a manner such that theheterologous DNA is expressed and a therapeutic product encoded therebyis produced. Alternatively, the heterologous DNA may in some mannermediate expression of DNA that encodes the therapeutic product; it mayencode a product, such as a peptide or RNA that in some manner mediates,directly or indirectly, expression of a therapeutic product. Genetictherapy may also be used to deliver nucleic acid encoding a gene productto replace a defective gene or supplement a gene product produced by themammal or the cell in which it is introduced. The introduced nucleicacid may encode a therapeutic compound, such as a growth factorinhibitor thereof, or a tumor necrosis factor or inhibitor thereof, suchas a receptor therefore, that is not normally produced in the mammalianhost or that is not produced in therapeutically effective amounts or ata therapeutically useful time. The heterologous DNA encoding thetherapeutic product may be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof.

As used herein, “heterologous nucleic acid sequence” is typically DNAthat encodes RNA and proteins that are not normally produced in vivo bythe cell in which it is expressed or that mediates or encodes mediatorsthat alter expression of endogenous DNA by affecting transcription,translation, or other regulatable biochemical processes. A heterologousnucleic acid sequence may also be referred to as foreign DNA. Any DNAthat one of skill in the art would recognize or consider as heterologousor foreign to the cell in which it is expressed is herein encompassed byheterologous DNA. Examples of heterologous DNA include, but are notlimited to, DNA that encodes traceable marker proteins, such as aprotein that confers drug resistance, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.Antibodies that are encoded by heterologous DNA may be secreted orexpressed on the surface of the cell in which the heterologous DNA hasbeen introduced.

As used herein the term “cardiomyopathy” refers to the deterioration ofthe function of the myocardium (i.e., the actual heart muscle) for anyreason. Subjects with cardiomyopathy are often at risk of arrhythmia,sudden cardiac death, or hospitalization or death due to heart failure.

As used herein, the term “ischemic cardiomyopathy” is a weakness in themuscle of the heart due to inadequate oxygen delivery to the myocardiumwith coronary artery disease being the most common cause.

As used herein the term “ischemic cardiac disease” refers to anycondition in which heart muscle is damaged or works inefficientlybecause of an absence or relative deficiency of its blood supply; mostoften caused by atherosclerosis, it includes angina pectoris, acutemyocardial infarction, chronic ischemic heart disease, and sudden death.

As used herein the term “myocardial infarction” refers to the damagingor death of an area of the heart muscle (myocardium) resulting from ablocked blood supply to that area.

As used herein the term “6-minute walk test” or “6MWT” refers to a testthat measures the distance that a patient can quickly walk on a flat,hard surface in a period of 6 minutes (the 6MWD). It evaluates theglobal and integrated responses of all the systems involved duringexercise, including the pulmonary and cardiovascular systems, systemiccirculation, peripheral circulation, blood, neuromuscular units, andmuscle metabolism. It does not provide specific information on thefunction of each of the different organs and systems involved inexercise or the mechanism of exercise limitation, as is possible withmaximal cardiopulmonary exercise testing. The self-paced 6MWT assessesthe submaximal level of functional capacity. (See for example, AM JRespir Crit. Care Med, Vol. 166. Pp 111-117 (2002))

As used herein “New York Heart Association (NYHA) functionalclassification” refers to a classification for the extent of heartfailure. It places patients in one of four categories based on how muchthey are limited during physical activity; the limitations/symptoms arein regards to normal breathing and varying degrees in shortness ofbreath and or angina pain:

NYHA Class Symptoms I No symptoms and no limitation in ordinary physicalactivity, e.g. shortness of breath when walking, climbing stairs etc. IIMild symptoms (mild shortness of breath and/or angina) and slightlimitation during ordinary activity. III Marked limitation in activitydue to symptoms, even during less- than-ordinary activity, e.g. walkingshort distances (20-100 m). Comfortable only at rest. IV Severelimitations. Experiences symptoms even while at rest. Mostly bedboundpatients.

This application relates to compositions and methods of treating acardiomyopathy in a subject that results in reduced and/or impairedmyocardial function. The cardiomyopathy treated by the compositions andmethods herein can include cardiomyopathies associated with a pulmonaryembolus, a venous thrombosis, a myocardial infarction, a transientischemic attack, a peripheral vascular disorder, atherosclerosis,ischemic cardiac disease and/or other myocardial injury or vasculardisease. The method of treating the cardiomyopathy can include locallyadministering (or locally delivering) to weakened myocardial tissue,ischemic myocardial tissue, and/or apoptotic myocardial tissue, such asthe peri-infarct region of a heart following myocardial infarction, anamount of stromal-cell derived factor-1 (SDF-1) that is effective tocause functional improvement in at least one of the followingparameters: left ventricular volume, left ventricular area, leftventricular dimension, cardiac function, 6-minute walk test (6MWT), orNew York Heart Association (NYHA) functional classification.

It was found using a porcine model of heart failure that mimics heartfailure in a human that functional improvement of ischemic myocardialtissue is dependent on the amount, dose, and/or delivery of SDF-1administered to the ischemic myocardial tissue and that the amount,dose, and/or delivery of SDF-1 to the ischemic myocardial tissue can beoptimized so that myocardial functional parameters, such as leftventricular volume, left ventricular area, left ventricular dimension,or cardiac function are substantially improved. As discussed below, insome aspects, the amount, concentration, and volume of SDF-1administered to the ischemic myocardial tissue can be controlled and/oroptimized to substantially improve the functional parameters (e.g., leftventricular volume, left ventricular area, left ventricular dimension,cardiac function, 6-minute walk test (6MWT), and/or New York HeartAssociation (NYHA) functional classification) while mitigating adverseside effects.

In one example, the SDF-1 can be administered directly or locally to aweakened region, an ischemic region, and/or peri-infarct region ofmyocardial tissue of a large mammal (e.g., pig or human) in which thereis a deterioration or worsening of a functional parameter of the heart,such as left ventricular volume, left ventricular area, left ventriculardimension, or cardiac function as a result of an ischemiccardiomyopathy, such as a myocardial infarction. The deterioration orworsening of the functional parameter can include, for example, anincrease in left ventricular end systolic volume, decrease in leftventricular ejection fraction, increase in wall motion score index,increase in left ventricular end diastolic length, increase in leftventricular end systolic length, increase in left ventricular enddiastolic area (e.g., mitral valve level and papillary muscle insertionlevel), increase in left ventricular end systolic area (e.g., mitralvalve level and papillary muscle insertion level), or increase in leftventricular end diastolic volume as measured using, for example, usingechocardiography.

In an aspect of the application, the amount of SDF-1 administered to theweakened region, ischemic region, and/or peri-infarct region of themyocardial tissue of the large mammal can be an amount effective toimprove at least one functional parameter of the myocardium, such as adecrease in left ventricular end systolic volume, increase in leftventricular ejection fraction, decrease in wall motion score index,decrease in left ventricular end diastolic length, decrease in leftventricular end systolic length, decrease in left ventricular enddiastolic area (e.g., mitral valve level and papillary muscle insertionlevel), decrease in left ventricular end systolic area (e.g., mitralvalve level and papillary muscle insertion level), or decrease in leftventricular end diastolic volume measured using, for example, usingechocardiography as well as improve the subject's 6-minute walk test(6MWT) or New York Heart Association (NYHA) functional classification.

In another aspect of the application, the amount of SDF-1 administeredto the weakened region, ischemic region, and/or peri-infarct region ofthe myocardial tissue of the large mammal with a cardiomyopathy iseffective to improve left ventricular end systolic volume in the mammalby at least about 10%, and more specifically at least about 15%, after30 days following administration as measured by echocardiography. Thepercent improvement is relative to each subject treated and is based onthe respective parameter measured prior to or at the time of therapeuticintervention or treatment.

In a further aspect of the application, the amount of SDF-1 administeredto the weakened region, ischemic region, and/or peri-infarct region ofthe myocardial tissue of the large mammal with a cardiomyopathy iseffective to improve left ventricular end systolic volume by at leastabout 10%, improve left ventricular ejection fraction by at least about10%, and improve wall motion score index by about 5%, after 30 daysfollowing administration as measured by echocardiography.

In a still further aspect of the application, the amount of SDF-1administered to the weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue of the large mammal with acardiomyopathy is effective to improve vasculogenesis of the weakenedregion, ischemic region, and/or peri-infarct region by at least 20%based on vessel density or an increase in cardiac perfusion measured bySPECT imaging. A 20% improvement in vasculogenesis has been shown to beclinically significant (Losordo Circulation 2002; 105:2012).

In a still further aspect of the application, the amount of SDF-1administered to the weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue of the large mammal with acardiomyopathy is effective to improve six minute walk distance at leastabout 30 meters or improve NYHA class by at least 1 class.

The SDF-1 described herein can be administered to the weakened region,the ischemic region, and/or peri-infarct region of the myocardial tissuefollowing tissue injury (e.g., myocardial infarction) to about hours,days, weeks, or months after onset of down-regulation of SDF-1. Theperiod of time that the SDF-1 is administered to the cells can comprisefrom about immediately after onset of the cardiomyopathy (e.g.,myocardial infarction) to about days, weeks, or months after the onsetof the ischemic disorder or tissue injury.

SDF-1 in accordance with the application that is administered to theweakened, ischemic, and/or a peri-infarct region of the myocardialtissue peri-infarct region can have an amino acid sequence that issubstantially similar to a native mammalian SDF-1 amino acid sequence.The amino acid sequence of a number of different mammalian SDF-1 proteinare known including human, mouse, and rat. The human and rat SDF-1 aminoacid sequences are at least about 92% identical (e.g., about 97%identical). SDF-1 can comprise two isoforms, SDF-1 alpha and SDF-1 beta,both of which are referred to herein as SDF-1 unless identifiedotherwise.

The SDF-1 can have an amino acid sequence substantially identical to SEQID NO: 1. The SDF-1 that is over-expressed can also have an amino acidsequence substantially similar to one of the foregoing mammalian SDF-1proteins. For example, the SDF-1 that is over-expressed can have anamino acid sequence substantially similar to SEQ ID NO: 2. SEQ ID NO: 2,which substantially comprises SEQ ID NO: 1, is the amino acid sequencefor human SDF-1 and is identified by GenBank Accession No. NP954637. TheSDF-1 that is over-expressed can also have an amino acid sequence thatis substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 includes theamino acid sequences for rat SDF and is identified by GenBank AccessionNo. AAF01066.

The SDF-1 in accordance with the application can also be a variant ofmammalian SDF-1, such as a fragment, analog and derivative of mammalianSDF-1. Such variants include, for example, a polypeptide encoded by anaturally occurring allelic variant of native SDF-1 gene (i.e., anaturally occurring nucleic acid that encodes a naturally occurringmammalian SDF-1 polypeptide), a polypeptide encoded by an alternativesplice form of a native SDF-1 gene, a polypeptide encoded by a homologor ortholog of a native SDF-1 gene, and a polypeptide encoded by anon-naturally occurring variant of a native SDF-1 gene.

SDF-1 variants have a peptide sequence that differs from a native SDF-1polypeptide in one or more amino acids. The peptide sequence of suchvariants can feature a deletion, addition, or substitution of one ormore amino acids of a SDF-1 variant. Amino acid insertions arepreferably of about 1 to 4 contiguous amino acids, and deletions arepreferably of about 1 to 10 contiguous amino acids. Variant SDF-1polypeptides substantially maintain a native SDF-1 functional activity.Examples of SDF-1 polypeptide variants can be made by expressing nucleicacid molecules that feature silent or conservative changes. One exampleof an SDF-1 variant is listed in U.S. Pat. No. 7,405,195, which isherein incorporated by reference in its entirety.

SDF-1 polypeptide fragments corresponding to one or more particularmotifs and/or domains or to arbitrary sizes, are within the scope ofthis application. Isolated peptidyl portions of SDF-1 can be obtained byscreening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. For example, anSDF-1 polypeptide may be arbitrarily divided into fragments of desiredlength with no overlap of the fragments, or preferably divided intooverlapping fragments of a desired length. The fragments can be producedrecombinantly and tested to identify those peptidyl fragments, which canfunction as agonists of native CXCR-4 polypeptides.

Variants of SDF-1 polypeptides can also include recombinant forms of theSDF-1 polypeptides. Recombinant polypeptides in some embodiments, inaddition to SDF-1 polypeptides, are encoded by a nucleic acid that canhave at least 70% sequence identity with the nucleic acid sequence of agene encoding a mammalian SDF-1.

SDF-1 variants can include agonistic forms of the protein thatconstitutively express the functional activities of native SDF-1. OtherSDF-1 variants can include those that are resistant to proteolyticcleavage, as for example, due to mutations, which alter protease targetsequences. Whether a change in the amino acid sequence of a peptideresults in a variant having one or more functional activities of anative SDF-1 can be readily determined by testing the variant for anative SDF-1 functional activity.

The SDF-1 nucleic acid that encodes the SDF-1 protein can be a native ornormative nucleic acid and be in the form of RNA or in the form of DNA(e.g., cDNA, genomic DNA, and synthetic DNA). The DNA can bedouble-stranded or single-stranded, and if single-stranded may be thecoding (sense) strand or non-coding (anti-sense) strand. The nucleicacid coding sequence that encodes SDF-1 may be substantially similar toa nucleotide sequence of the SDF-1 gene, such as nucleotide sequenceshown in SEQ ID NO: 4 and SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5comprise, respectively, the nucleic acid sequences for human SDF-1 andrat SDF-1 and are substantially similar to the nucleic sequences ofGenBank Accession No. NM199168 and GenBank Accession No. AF189724.

The nucleic acid coding sequence for SDF-1 can also be a differentcoding sequence which, as a result of the redundancy or degeneracy ofthe genetic code, encodes the same polypeptide as SEQ ID NO: 1, SEQ IDNO: 2, and SEQ ID NO: 3.

Other nucleic acid molecules that encode SDF-1 are variants of a nativeSDF-1, such as those that encode fragments, analogs and derivatives ofnative SDF-1. Such variants may be, for example, a naturally occurringallelic variant of a native SDF-1 gene, a homolog or ortholog of anative SDF-1 gene, or a non-naturally occurring variant of a nativeSDF-1 gene. These variants have a nucleotide sequence that differs froma native SDF-1 gene in one or more bases. For example, the nucleotidesequence of such variants can feature a deletion, addition, orsubstitution of one or more nucleotides of a native SDF-1 gene. Nucleicacid insertions are preferably of about 1 to 10 contiguous nucleotides,and deletions are preferably of about 1 to 10 contiguous nucleotides.

In other applications, variant SDF-1 displaying substantial changes instructure can be generated by making nucleotide substitutions that causeless than conservative changes in the encoded polypeptide. Examples ofsuch nucleotide substitutions are those that cause changes in (a) thestructure of the polypeptide backbone; (b) the charge or hydrophobicityof the polypeptide; or (c) the bulk of an amino acid side chain.Nucleotide substitutions generally expected to produce the greatestchanges in protein properties are those that cause non-conservativechanges in codons. Examples of codon changes that are likely to causemajor changes in protein structure are those that cause substitution of(a) a hydrophilic residue e.g., serine or threonine), for (or by) ahydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine oralanine); (b) a cysteine or proline for (or by) any other residue; (c) aresidue having an electropositive side chain (e.g., lysine, arginine, orhistidine), for (or by) an electronegative residue (e.g., glutamine oraspartine); or (d) a residue having a bulky side chain (e.g.,phenylalanine), for (or by) one not having a side chain, (e.g.,glycine).

Naturally occurring allelic variants of a native SDF-1 gene are nucleicacids isolated from mammalian tissue that have at least 70% sequenceidentity with a native SDF-1 gene, and encode polypeptides havingstructural similarity to a native SDF-1 polypeptide. Homologs of anative SDF-1 gene are nucleic acids isolated from other species thathave at least 70% sequence identity with the native gene, and encodepolypeptides having structural similarity to a native SDF-1 polypeptide.Public and/or proprietary nucleic acid databases can be searched toidentify other nucleic acid molecules having a high percent (e.g., 70%or more) sequence identity to a native SDF-1 gene.

Non-naturally occurring SDF-1 gene variants are nucleic acids that donot occur in nature (e.g., are made by the hand of man), have at least70% sequence identity with a native SDF-1 gene, and encode polypeptideshaving structural similarity to a native SDF-1 polypeptide. Examples ofnon-naturally occurring SDF-1 gene variants are those that encode afragment of a native SDF-1 protein, those that hybridize to a nativeSDF-1 gene or a complement of to a native SDF-1 gene under stringentconditions, and those that share at least 65% sequence identity with anative SDF-1 gene or a complement of a native SDF-1 gene.

Nucleic acids encoding fragments of a native SDF-1 gene in someembodiments are those that encode amino acid residues of native SDF-1.Shorter oligonucleotides that encode or hybridize with nucleic acidsthat encode fragments of native SDF-1 can be used as probes, primers, orantisense molecules. Longer polynucleotides that encode or hybridizewith nucleic acids that encode fragments of a native SDF-1 can also beused in various aspects of the application. Nucleic acids encodingfragments of a native SDF-1 can be made by enzymatic digestion (e.g.,using a restriction enzyme) or chemical degradation of the full-lengthnative SDF-1 gene or variants thereof.

Nucleic acids that hybridize under stringent conditions to one of theforegoing nucleic acids can also be used herein. For example, suchnucleic acids can be those that hybridize to one of the foregoingnucleic acids under low stringency conditions, moderate stringencyconditions, or high stringency conditions.

Nucleic acid molecules encoding a SDF-1 fusion protein may also be usedin some embodiments. Such nucleic acids can be made by preparing aconstruct (e.g., an expression vector) that expresses a SDF-1 fusionprotein when introduced into a suitable target cell. For example, such aconstruct can be made by ligating a first polynucleotide encoding aSDF-1 protein fused in frame with a second polynucleotide encodinganother protein such that expression of the construct in a suitableexpression system yields a fusion protein.

The nucleic acids encoding SDF-1 can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The nucleic acids described hereinmay additionally include other appended groups such as peptides (e.g.,for targeting target cell receptors in vivo), or agents facilitatingtransport across the cell membrane, hybridization-triggered cleavage. Tothis end, the nucleic acids may be conjugated to another molecule,(e.g., a peptide), hybridization triggered cross-linking agent,transport agent, hybridization-triggered cleavage agent, etc.

The SDF-1 can be delivered to the weakened, ischemic, and/orperi-infarct region of the myocardial tissue by administering an SDF-1protein to the weakened, ischemic, and/or peri-infarct region, or byintroducing an agent into cells of the weakened region, ischemic region,and/or peri-infarct region of the myocardial tissue that causes,increases, and/or upregulates expression of SDF-1 (i.e., SDF-1 agent).The SDF-1 protein expressed from the cells can be an expression productof a genetically modified cell.

The agent that causes, increases, and/or upregulates expression of SDF-1can comprise natural or synthetic nucleic acids as described herein thatare incorporated into recombinant nucleic acid constructs, typically DNAconstructs, capable of introduction into and replication in the cells ofthe myocardial tissue. Such a construct can include a replication systemand sequences that are capable of transcription and translation of apolypeptide-encoding sequence in a given cell.

One method of introducing the agent into a target cell involves usinggene therapy. Gene therapy in some embodiments of the application can beused to express SDF-1 protein from a cell of the weakened region,ischemic region, and/or peri-infarct region of the myocardial tissue invivo.

In an aspect of the application, the gene therapy can use a vectorincluding a nucleotide encoding an SDF-1 protein. A “vector” (sometimesreferred to as gene delivery or gene transfer “vehicle”) refers to amacromolecule or complex of molecules comprising a polynucleotide to bedelivered to a target cell, either in vitro or in vivo. Thepolynucleotide to be delivered may comprise a coding sequence ofinterest in gene therapy. Vectors include, for example, viral vectors(such as adenoviruses (‘Ad’), adeno-associated viruses (AAV), andretroviruses), non-viral vectors, liposomes, and other lipid-containingcomplexes, and other macromolecular complexes capable of mediatingdelivery of a polynucleotide to a target cell.

Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities.

Selectable markers can be positive, negative or bifunctional. Positiveselectable markers allow selection for cells carrying the marker,whereas negative selectable markers allow cells carrying the marker tobe selectively eliminated. A variety of such marker genes have beendescribed, including bifunctional (i.e., positive/negative) markers(see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton,S., WO 94/28143, published Dec. 8, 1994). Such marker genes can providean added measure of control that can be advantageous in gene therapycontexts. A large variety of such vectors are known in the art and aregenerally available.

Vectors for use herein include viral vectors, lipid based vectors andother nonviral vectors that are capable of delivering a nucleotide tothe cells of weakened region, ischemic region, and/or peri-infarctregion of the myocardial tissue. The vector can be a targeted vector,especially a targeted vector that preferentially binds to the cells ofweakened region, ischemic region, and/or peri-infarct region of themyocardial tissue. Viral vectors for use in the methods herein caninclude those that exhibit low toxicity to the cells of weakened region,ischemic region, and/or peri-infarct region of the myocardial tissue andinduce production of therapeutically useful quantities of SDF-1 proteinin a tissue-specific manner.

Examples of viral vectors are those derived from adenovirus (Ad) oradeno-associated virus (AAV). Both human and non-human viral vectors canbe used and the recombinant viral vector can be replication-defective inhumans. Where the vector is an adenovirus, the vector can comprise apolynucleotide having a promoter operably linked to a gene encoding theSDF-1 protein and is replication-defective in humans.

Other viral vectors that can be used in accordance with method of theapplication include herpes simplex virus (HSV)-based vectors. HSVvectors deleted of one or more immediate early genes (1E) areadvantageous because they are generally non-cytotoxic, persist in astate similar to latency in the target cell, and afford efficient targetcell transduction. Recombinant HSV vectors can incorporate approximately30 kb of heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, might alsobe used in some embodiments of the application. For example, retroviralvectors may be based on murine leukemia virus (MLV). See, e.g., Hu andPathak, Pharmacal. Rev. 52:493-511, 2000 and Pong et al., Crit. Rev.Ther. Drug Carrier Syst. 17:1-60, 2000. MLV-based vectors may contain upto 8 kb of heterologous (therapeutic) DNA in place of the viral genes.The heterologous DNA may include a tissue-specific promoter and an SDF-1nucleic acid. In methods of delivery to cells proximate the wound, itmay also encode a ligand to a tissue specific receptor.

Additional retroviral vectors that might be used arereplication-defective lentivirus-based vectors, including humanimmunodeficiency (HIV)-based vectors. See, e.g., Vigna and Naldini, J.Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Viral. 72:8150-8157,1998. Lentiviral vectors are advantageous in that they are capable ofinfecting both actively dividing and non-dividing cells. They are alsohighly efficient at transducing human epithelial cells.

Lentiviral vectors for use in the methods herein may be derived fromhuman and non-human (including SIV) lentiviruses. Examples of lentiviralvectors include nucleic acid sequences required for vector propagationas well as a tissue-specific promoter operably linked to a SDF-1 gene.These former may include the viral LTRs, a primer binding site, apolypurine tract, att sites, and an encapsidation site.

A lentiviral vector may be packaged into any suitable lentiviral capsid.The substitution of one particle protein with another from a differentvirus is referred to as “pseudotyping”. The vector capsid may containviral envelope proteins from other viruses, including murine leukemiavirus (MLV) or vesicular stomatitis virus (VSV). The use of the VSVG-protein yields a high vector titer and results in greater stability ofthe vector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus(SFV) and sindbis virus (SIN) might also be used herein. Use ofalphaviruses is described in Lundstrom, K., Intervirology 43:247-257,2000 and Perri et al., Journal of Virology 74:9802-9807, 2000.

Recombinant, replication-defective alphavirus vectors are advantageousbecause they are capable of high-level heterologous (therapeutic) geneexpression, and can infect a wide target cell range. Alphavirusreplicons may be targeted to specific cell types by displaying on theirvirion surface a functional heterologous ligand or binding domain thatwould allow selective binding to target cells expressing a cognatebinding partner. Alphavirus replicons may establish latency, andtherefore long-term heterologous nucleic acid expression in a targetcell. The replicons may also exhibit transient heterologous nucleic acidexpression in the target cell.

In many of the viral vectors compatible with methods of the application,more than one promoter can be included in the vector to allow more thanone heterologous gene to be expressed by the vector. Further, the vectorcan comprise a sequence which encodes a signal peptide or other moietywhich facilitates the expression of a SDF-1 gene product from the targetcell.

To combine advantageous properties of two viral vector systems, hybridviral vectors may be used to deliver a SDF-1 nucleic acid to a targettissue. Standard techniques for the construction of hybrid vectors arewell-known to those skilled in the art. Such techniques can be found,for example, in Sambrook, et al., In Molecular Cloning: A laboratorymanual. Cold Spring Harbor, N.Y. or any number of laboratory manualsthat discuss recombinant DNA technology. Double-stranded AAV genomes inadenoviral capsids containing a combination of AAV and adenoviral ITRsmay be used to transduce cells. In another variation, an AAV vector maybe placed into a “gutless”, “helper-dependent” or “high-capacity”adenoviral vector. Adenovirus/AAV hybrid vectors are discussed in Lieberet al., J. Viral. 73:9314-9324, 1999. Retrovirus/adenovirus hybridvectors are discussed in Zheng et al., Nature Biotechnol. 18:176-186,2000. Retroviral genomes contained within an adenovirus may integratewithin the target cell genome and effect stable SDF-1 gene expression.

Other nucleotide sequence elements which facilitate expression of theSDF-1 gene and cloning of the vector are further contemplated. Forexample, the presence of enhancers upstream of the promoter orterminators downstream of the coding region, for example, can facilitateexpression.

In accordance with another aspect of the application, a tissue-specificpromoter, can be fused to a SDF-1 gene. By fusing such tissue specificpromoter within the adenoviral construct, transgene expression islimited to a particular tissue. The efficacy of gene expression anddegree of specificity provided by tissue specific promoters can bedetermined, using the recombinant adenoviral system described herein.

In addition to viral vector-based methods, non-viral methods may also beused to introduce a SDF-1 nucleic acid into a target cell. A review ofnon-viral methods of gene delivery is provided in Nishikawa and Huang,Human Gene Ther. 12:861-870, 2001. An example of a non-viral genedelivery method according to the invention employs plasmid DNA tointroduce a SDF-1 nucleic acid into a cell. Plasmid-based gene deliverymethods are generally known in the art. In one example, the plasmidvector can have a structure as shown schematically in FIG. 7. Theplasmid vector of FIG. 7 includes a CMV enhancer and CMV promoterupstream of an SDF-1α cDNA (RNA) sequence.

Optionally, synthetic gene transfer molecules can be designed to formmultimolecular aggregates with plasmid SDF-1 DNA. These aggregates canbe designed to bind to cells of weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue. Cationic amphiphiles,including lipopolyamines and cationic lipids, may be used to providereceptor-independent SDF-1 nucleic acid transfer into target cells(e.g., cardiomyocytes). In addition, preformed cationic liposomes orcationic lipids may be mixed with plasmid DNA to generatecell-transfecting complexes. Methods involving cationic lipidformulations are reviewed in Feigner et al., Ann. N.Y. Acad. Sci.772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev.20:221-266, 1996. For gene delivery, DNA may also be coupled to anamphipathic cationic peptide (Fominaya et al., J. Gene Med. 2:455-464,2000).

Methods that involve both viral and non-viral based components may beused herein. For example, an Epstein Barr virus (EBV)-based plasmid fortherapeutic gene delivery is described in Cui et al., Gene Therapy8:1508-1513, 2001. Additionally, a method involving aDNA/ligand/polycationic adjunct coupled to an adenovirus is described inCuriel, D. T., Nat. Immun. 13:141-164, 1994.

Additionally, the SDF-1 nucleic acid can be introduced into the targetcell by transfecting the target cells using electroporation techniques.Electroporation techniques are well known and can be used to facilitatetransfection of cells using plasmid DNA.

Vectors that encode the expression of SDF-1 can be delivered to thetarget cell in the form of an injectable preparation containing apharmaceutically acceptable carrier, such as saline, as necessary. Otherpharmaceutical carriers, formulations and dosages can also be used inaccordance with the present invention.

In one aspect of the invention, the vector can comprise an SDF-1plasmid, such as for example in FIG. 7. SDF-1 plasmid can be deliveredto cells of the weakened region, ischemic region, and/or peri-infarctregion of the myocardial tissue by direct injection of the SDF-1 plasmidvector into the weakened region, ischemic region, and/or peri-infarctregion of the myocardial tissue at an amount effective to improve atleast one myocardial functional parameters, such as left ventricularvolume, left ventricular area, left ventricular dimension, or cardiacfunction as well as improve the subject's 6-minute walk test (6MWT) orNew York Heart Association (NYHA) functional classification. Byinjecting the vector directly into or about the periphery of theweakened region, ischemic region, and/or peri-infarct region of themyocardial tissue, it is possible to target the vector transfectionrather effectively, and to minimize loss of the recombinant vectors.This type of injection enables local transfection of a desired number ofcells, especially about the weakened region, ischemic region, and/orperi-infarct region of the myocardial tissue, thereby maximizingtherapeutic efficacy of gene transfer, and minimizing the possibility ofan inflammatory response to viral proteins.

In an aspect of the application, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in multipleinjections of a solution of SDF-1 expressing plasmid DNA with eachinjection comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1plasmid/solution. In one example, the SDF-1 plasmid can be administeredto the weakened, ischemic, and/or peri-infarct region in at least about10 injections, at least about 15 injections, or at least about 20injections. Multiple injections of the SDF-1 plasmid to the weakened,ischemic, and/or peri-infarct region allows a greater area and/or numberof cells of the weakened, ischemic, and/or peri-infarct region to betreated.

Each injection administered to the weakened, ischemic, and/orperi-infarct region can have a volume of at least about 0.2 ml. Thetotal volume of solution that includes the amount of SDF-1 plasmidadministered to the weakened, ischemic, and/or peri-infarct region thatcan improve at least one functional parameter of the heart is at leastabout 10 ml.

In one example, the SDF-1 plasmid can be administered to the weakened,ischemic, and/or peri-infarct region in at least about 10 injections.Each injection administered to the weakened, ischemic, and/orperi-infarct region can have a volume of at least about 0.2 ml. TheSDF-1 can be expressed in the weakened, ischemic, and/or peri-infarctregion for greater than about three days.

For example, each injection of solution including SDF-1 expressingplasmid can have an injection volume of at least about 0.2 ml and anSDF-1 plasmid concentration per injection of about 0.33 mg/ml to about 5mg/ml. In another aspect of the application, at least one functionalparameter of the of the heart can be improved by injecting the SDF-1plasmid into the weakened, ischemic, and/or peri-infarct region of theheart at an injection volume per site of at least about 0.2 ml, in atleast about 10 injection sites, and at an SDF-1 plasmid concentrationper injection of about 0.33 mg/ml to about 5 mg/ml.

It was found in a porcine model of congestive heart failure thatinjections of a solution of SDF-1 plasmid having concentration of lessabout 0.33 mg/ml or greater than about 5 mg/ml and an injection volumeper injection site less than about 0.2 ml to a porcine model of heartfailure resulted in little if any functional improvement of the leftventricular volume, left ventricular area, left ventricular dimension,or cardiac function of the treated heart.

In another aspect of the application, the amount of SDF-1 plasmidadministered to the weakened, ischemic, and/or peri-infarct region thatcan improve at least one functional parameter of the heart is greaterthan about 4 mg and less than about 100 mg per therapeutic intervention.The amount of SDF-1 plasmid administered by therapeutic interventionherein refers to the total SDF-1 plasmid administered to the subjectduring a therapeutic procedure designed to affect or elicit atherapeutic effect. This can include the total SDF-1 plasmidadministered in single injection for a particular therapeuticintervention or the total SDF-1 plasmid that is administered by multipleinjections for a therapeutic intervention. It was found in a porcinemodel of congestive heart failure that administration of about 4 mgSDF-1 plasmid DNA via direct injection of the SDF-1 plasmid to the heartresulted in no functional improvement of the left ventricular volume,left ventricular area, left ventricular dimension, or cardiac functionof the treated heart. Moreover, administration of about 100 mg of SDF-1plasmid DNA via direct injection of the SDF-1 plasmid to the heartresulted in no functional improvement of the left ventricular volume,left ventricular area, left ventricular dimension, or cardiac functionof the treated heart.

In some aspects of the application, the SDF-1 can be expressed at atherapeutically effective amount or dose in the weakened, ischemic,and/or peri-infarct region after transfection with the SDF-1 plasmidvector for greater than about three days. Expression of SDF-1 at atherapeutically effective dose or amount for greater three days canprovide a therapeutic effect to weakened, ischemic, and/or peri-infarctregion. Advantageously, the SDF-1 can be expressed in the weakened,ischemic, and/or peri-infarct region after transfection with the SDF-1plasmid vector at a therapeutically effective amount for less than about90 days to mitigate potentially chronic and/or cytotoxic effects thatmay inhibit the therapeutic efficacy of the administration of the SDF-1to the subject.

It will be appreciated that the amount, volume, concentration, and/ordosage of SDF-1 plasmid that is administered to any one animal or humandepends on many factors, including the subject's size, body surfacearea, age, the particular composition to be administered, sex, time androute of administration, general health, and other drugs beingadministered concurrently. Specific variations of the above notedamounts, volumes, concentrations, and/or dosages of SDF-1 plasmid canreadily be determined by one skilled in the art using the experimentalmethods described below.

In another aspect of the application, the SDF-1 plasmid can beadministered by direct injection using catheterization, such asendo-ventricular catheterization or intra-myocardial catheterization. Inone example, a deflectable guide catheter device can be advanced to aleft ventricle retrograde across the aortic valve. Once the device ispositioned in the left ventricle, SDF-1 plasmid can be injected into theperi-infarct region (both septal and lateral aspect) area of the leftventricle. Typically, 1.0 ml of SDF-1 plasmid solution can be injectionover a period of time of about 60 seconds. The subject being treated canreceive at least about 10 injection (e.g., about 15 to about 20injections in total).

The myocardial tissue of the subject can be imaged prior toadministration of the SDF-1 plasmid to define the area of weakened,ischemic, and/or peri-infarct region prior to administration of theSDF-1 plasmid. Defining the weakened, ischemic, and/or peri-infarctregion by imaging allows for more accurate intervention and targeting ofthe SDF-1 plasmid to the weakened, ischemic, and/or peri-infarct region.The imaging technique used to define the weakened, ischemic, and/orperi-infarct region of the myocardial tissue can include any knowncardia-imaging technique. Such imaging techniques can include, forexample, at least one of echocardiography, magnetic resonance imaging,coronary angiogram, electroanatomical mapping, or fluoroscopy. It willbe appreciated that other imaging techniques that can define theweakened, ischemic, and/or peri-infarct region can also be used.

Optionally, other agents besides SDF-1 nucleic acids (e.g., SDF-1plasmids) can be introduced into the weakened, ischemic, and/orperi-infarct region of the myocardial tissue to promote expression ofSDF-1 from cells of the weakened, ischemic, and/or peri-infarct region.For example, agents that increase the transcription of a gene encodingSDF-1 increase the translation of an mRNA encoding SDF-1, and/or thosethat decrease the degradation of an mRNA encoding SDF-1 could be used toincrease SDF-1 protein levels. Increasing the rate of transcription froma gene within a cell can be accomplished by introducing an exogenouspromoter upstream of the gene encoding SDF-1. Enhancer elements, whichfacilitate expression of a heterologous gene, may also be employed.

Other agents can include other proteins, chemokines, and cytokines, thatwhen administered to the target cells can upregulate expression of SDF-1by the weakened, ischemic, and/or peri-infarct region of the myocardialtissue. Such agents can include, for example: insulin-like growth factor(IGF)-1, which was shown to upregulate expression of SDF-1 whenadministered to mesenchymal stem cells (MSCs) (Circ. Res. 2008, November21; 103(11):1300-98); sonic hedgehog (Shh), which was shown toupregulate expression of SDF-1 when administered to adult fibroblasts(Nature Medicine, Volume 11, Number 11, November 23); transforminggrowth factor β (TGF-β); which was shown to upregulate expression ofSDF-1 when administered to human peritoneal mesothelial cells (HPMCs);IL-1β, PDGF, VEGF, TNF-a, and PTH, which are shown to upregulateexpression of SDF-1, when administered to primary human osteoblasts(HOBs) mixed marrow stromal cells (BMSCs), and human osteoblast-likecell lines (Bone, 2006, April; 38(4): 497-508); thymosin (34, which wasshown to upregulate expression when administered to bone marrow cells(BMCs) (Curr. Pharm. Des. 2007; 13(31):3245-51; and hypoxia induciblefactor 1α (HIF-1), which was shown to upregulate expression of SDF-1when administered to bone marrow derived progenitor cells (Cardiovasc.Res. 2008, E. Pub.). These agents can be used to treat specificcardiomyopathies where such cells capable of upregulating expression ofSDF-1 with respect to the specific cytokine are present or administered.

The SDF-1 protein or agent, which causes increases, and/or upregulatesexpression of SDF-1, can be administered to the weakened, ischemic,and/or peri-infarct region of the myocardial tissue neat or in apharmaceutical composition. The pharmaceutical composition can providelocalized release of the SDF-1 or agent to the cells of the weakened,ischemic, and/or peri-infarct region being treated. Pharmaceuticalcompositions in accordance with the application will generally includean amount of SDF-1 or agent admixed with an acceptable pharmaceuticaldiluent or excipient, such as a sterile aqueous solution, to give arange of final concentrations, depending on the intended use. Thetechniques of preparation are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. Moreover,for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

The pharmaceutical composition can be in a unit dosage injectable form(e.g., solution, suspension, and/or emulsion). Examples ofpharmaceutical formulations that can be used for injection includesterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, liquidpolyethylene glycol, and the like), dextrose, saline, orphosphatebuffered saline, suitable mixtures thereof and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions.

Additionally, various additives, which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tomethods described herein, however, any vehicle, diluent, or additiveused would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the methods described herein in therequired amount of the appropriate solvent with various amounts of theother ingredients, as desired.

Pharmaceutical “slow release” capsules or “sustained release”compositions or preparations may be used and are generally applicable.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliver the SDF-1 oragent. The slow release formulations are typically implanted in thevicinity of the weakened, ischemic, and/or peri-infarct region of themyocardial tissue.

Examples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the SDF-1 or agent,which matrices are in the form of shaped articles, e.g., films ormicrocapsule. Examples of sustained-release matrices include polyesters;hydrogels, for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol); polylactides, e.g., U.S. Pat. No. 3,773,919;copolymers of L-glutamic acid and y ethyl-L-glutamate; non-degradableethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers,such as the LUPRON DEPOT (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate); andpoly-D-(−)-3-hydroxybutyric acid.

While polymers, such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated, SDF-1 orthe agent can remain in the body for a long time, and may denature oraggregate as a result of exposure to moisture at 37° C., thus reducingbiological activity and/or changing immunogenicity. Rational strategiesare available for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism involves intermolecular S—S bondformation through thio-disulfide interchange, stabilization is achievedby modifying sulfhydryl residues, lyophilizing from acidic solutions,controlling moisture content, using appropriate additives, developingspecific polymer matrix compositions, and the like.

In certain embodiments, liposomes and/or nanoparticles may also beemployed with the SDF-1 or agent. The formation and use of liposomes isgenerally known to those of skill in the art, as summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLV s results in theformation of small unilamellar vesicles (SUV s) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios, the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the methods, and such particles are easily made.

For preparing pharmaceutical compositions from the compounds of theapplication, pharmaceutically acceptable carriers can be in any form(e.g., solids, liquids, gels, etc.). A solid carrier can be one or moresubstances, which may also act as diluents, flavoring agents, binders,preservatives, and/or an encapsulating material.

Critical Limb Ischemia

This application additionally relates to a method of treating criticallimb ischemia in a subject. The method includes administering, JVS-100by direct intramuscular injection to the upper leg (quadriceps muscles)and lower leg (primarily gastrocnemius muscle) using multiple injectionsites. JVS-100 is the sterile biological product, composed of the nakedDNA plasmid encoding human SDF-1 cDNA (ACL-01110Sk) and 5% dextrose.

Critical limb ischemia (CLI) represents the most advanced stage ofatherosclerotic, lower extremity peripheral vascular disease (PVD) andis associated with high rates of cardiovascular morbidity, mortality,and major amputation. The incidence of CLI is estimated to be 125,000 to250,000 patients per year in the United States and is expected to growas the population ages. PVD prevalence increases dramatically with ageand affects approximately 20% of Americans age 65 and older. The currentstandard of care for individuals with CLI includes lower extremityrevascularization, either through open peripheral surgical procedures,endovascular techniques, or lower extremity amputation (i.e. ifrevascularization has failed or is not feasible). The 1-year mortalityrate of patients with CLI is 25% and may be as high as 45% in those whohave undergone amputation. Despite advanced techniques in vascular andsurgical procedures, a considerable proportion of patients with CLI arenot suitable for revascularization. Of these patients, 30% will requiremajor amputation and 23% will die within 3 months. Strategies to openblocked vessels or stimulate angiogenesis are under activeinvestigation.

Therapeutic angiogenesis, first evaluated by Dr. Jeffrey Isner in a 71year-old patient with severe PVD and toe gangrene in 1994, is a strategyfor the treatment of CLI that utilizes angiogenic or vasculogenic growthfactors. Genes to encode these growth factors are injected into ischemictissue to promote neovascularization in an attempt to increase perfusionto ischemic tissues through various mechanisms of action. In this study,human plasmid phVEGF165 was applied by balloon angioplasty to the distalpopliteal artery. Functional and angiographic parameters improved within12 weeks, and spider angiomata and edema developed unilaterally in theaffected limb, suggesting the treatment had a local angiogenic effect.This pioneer experiment suggested that experimental CLI therapies thatattempt to increase expression of angiogenic growth factors in ischemictissue may be beneficial to provide patients with poor surgical outcomesthe angiogenic potential to restore function and preserve the limb.Recent studies have demonstrated that chemokines that stimulateangiogenesis may be a critical component of therapies directed atretaining and restoring function in the limbs of patients with criticallimb ischemia.

Non-viral gene delivery, or the application of naked plasmid DNA toexpress a therapeutic protein at a specific site, is a simple deliverymethod that has been tested clinically in ischemic patients for over 15years. The safety profile of non-viral gene delivery is also attractivewhen compared to viral vector therapy delivery because it does notproduce a significant inflammatory response elicited by viral vectordelivery. A substantial body of literature, both preclinical andclinical, has demonstrated that non-viral delivery of therapeutic genesis safe and effective in disease models such as critical limb ischemia,cardiac myopathy and wound healing. In particular, plasmid DNA encodingfibroblast growth factor (FGF), vascular endothelial growth factor(VEGF), hepatocyte growth factor (HGF) and have been used to treatpatients with CLI in attempts to increase collateral blood flow to areascompromised by poor or damaged vasculature. Importantly, clinical use ofnon-viral FGF and VEGF gene therapy has been shown to be safe with nosignificant safety concerns reported to date. In several Phase I and IIstudies, patients with unreconstructable severe PVD with rest pain ortissue necrosis underwent treatment with intramuscular injection ofnon-viral FGF (NV 1 FGF). Increasing single (up to 16 mg) and repeated(2× up to 8 mg) doses of NV1FGF were injected into the ischemic thighand calf using naked plasmid DNA. NV1FGF was well tolerated, and after6-month follow up of 38 patients, a significant reduction in pain scaleand ischemic ulcer size, as well as increase in TcPO₂ compared tobaseline values was observed. Large-scale phase III trials examining theuse of naked plasmid DNA to treat CLI, such as the multicenterdouble-blind, placebo-controlled trial evaluating efficacy and safety ofNV1FGF in CLI patients with skin lesions the TAMARIS trial, areunderway, which will include 490 patients assigned to placebo orintramuscular injection of NV1FGF.

These non-viral gene therapies have shown no safety concerns to date andNVIFGF appears to provide benefit in subjects with CLI based on Phase IIclinical data. This, coupled with our previous preclinical work withACRX-100 treatment of ischemic heart failure which has demonstratedimprovement in vasculogenesis and cardiac function with no safetyissues, led us to hypothesize that ACRX-100 will be safe, improvevasculogenesis and ultimately provide clinical benefit in Critical LimbIschemia patients.

It has been determined that SDF-1 is upregulated in multiple tissuesafter injury and is expressed for a period of 4-5 days. Multiple groupshave demonstrated the therapeutic potential of SDF-1 therapy in a broadrange of diseases, including: ischemic myopathy, peripheral vasculardisease, wound healing, critical limb ischemia, and diabetes. SDF-1 is astrong chemoattractant of stem cells and progenitor cells that promotetissue preservation and blood vessel development. Together, thesereports point to a conserved pathway and mechanism of action throughwhich SDF-1 may promote repair and restore function after tissue injury.This led the inventors to develop ACRX-100 (a non-viral, naked DNAplasmid encoding SDF-1 in dextrose solution) for treatment of ischemiccardiovascular disease. In a GLP safety and toxicology study, ACRX-100demonstrated functional benefit up to 30 mg and safety up to 100 mg in aporcine model of heart failure. The inventors are currently enrolling amulti-center, open-label, dose-escalation Phase I clinical trial usingACRX-100 to treat patients with ischemic heart failure.

The inventors' studies demonstrated that ACRX-100 significantlyincreased vasculogenesis in the heart, consistent with studies fromseveral groups. These studies suggest re-establishing stem cell homingto chronically damaged tissue in critical limb ischemia may reinitiatetissue repair and potentially improve blood flow. Further studies havegone on to show that direct intramuscular injections of ACRX-100 into anischemic hindlimb of a rabbit promoted revascularization of the tissuecompared to control treated animals, suggesting that ACRX-100 is apromising therapeutic candidate for treating patients with critical limbischemia (CLI). The incidence of CLI is estimated to be 125,000 to250,000 patients per year in the United States and is expected to growas the population ages. The current standard of care for CLI patientsincludes lower extremity revascularization, either through openperipheral surgical procedures, endovascular techniques, or lowerextremity amputation (i.e., if revascularization has failed or is notfeasible). The 1-year mortality rate of patients with CLI is 25% and maybe as high as 45% in those who have undergone amputation. Despiteadvanced techniques in vascular and surgical procedures, a considerableproportion of patients with CLI are not suitable for revascularization.

ACL-01110SK is naked DNA plasmid encoding human SDF-1 cDNA. ACRX-100 isthe sterile biological product, composed of ACL-01110SK and 5% dextrose.

ACL-01110SK (FIG. 7) is a naked DNA plasmid designed to express humanSDF-1 in mammalian tissue. The plasmid backbone consists of the ColE1origin and the kanamycin resistance marker. SDF-1 transgene expressionis driven by the CMV enhancer/promoter, CMV-intron A and the RU5translational enhancer. Efficient polyadenylation is ensured by theincorporation of the bovine growth hormone polyA signal sequence. Thisis the same plasmid and formulation used for the treatment of patientswith heart failure. The inventors are developing ACRX-100 for thetreatment of patients with critical limb ischemia. ACRX-100 isformulated for direct intramuscular injection. The planned dosingregimen is comprised of single or multiple dose administration to theupper leg (quadriceps muscles) and lower leg (primarily gastrocnemiusmuscle) using multiple injection sites.

SDF-1 (a.k.a. CXCL12) is a naturally-occurring chemokine that is rapidlyupregulated in response to tissue injury. SDF-1 induction stimulates anumber of protective anti-inflammatory pathways, causes the downregulation of pro-inflammatory mediators (such as MMP-9 and IL-8), andcan protect cells from apoptosis by inhibiting caspase-mediatedactivation of Akt. Furthermore, SDF-1 is a strong chemoattractant ofendogenous organ specific and bone marrow derived stem cells andprogenitor cells to the site of tissue damage, which promotes tissuepreservation and blood vessel development. Previous studies havedemonstrated that SDF-1 expression is increased at the site of aninjury, but expression lasts for less than a week, and therefore theinduced stem cell homing response quickly fades. This short duration ofSDF-1 expression reduces the potential for tissue repair but suggeststhat therapeutic interventions that prolong or re-introduce the abilityof SDF-1 to stimulate the stem cell homing process may be beneficial forpatients that have damaged tissue. Based on this hypothesis, Dr. Penn'slaboratory demonstrated that re-introducing SDF-1 by injecting cardiacfibroblasts overexpressing SDF-1 months after MI resulted inre-establishment of the homing of bone marrow derived and endogenousorgan specific stem cells to the heart, growth of new blood vesselswithin the injured tissue, and a >80% increase in heart function.Skeletal myoblasts overexpressing SDF-1 injected eight weeks post-MIalso significantly improved cardiac function. Furthermore, SDF-1improved cardiac function by enhancing recruitment of circulating stemcells to injured tissue and causing formation of new blood vessels toincrease perfusion to the injured region. These effects, along withsubstantial preservation of myocardium, were demonstrated when SDF-1overexpressing MSCs were delivered to rats following acute MI leading toa 240% increase in cardiac function. This biologic response has beenconserved in a number of organ systems in response to ischemic injury.

The benefit of SDF-1 treatment observed by the inventors has beenvalidated by recent work in other independent laboratories. SDF-1 hasimproved cardiac function in ischemic cardiomyopathy when it has beendelivered by: nanofiber-embedded protein in post-acute MI rats,recombinant protein via a fibrin patch in post-MI mice, or directintramyocardial injection of protein in post-acute MI mice.SDF-1-encoding plasmid injected into the MI border zone has been shownto attract circulating stem cells to the MI border region. Similarly,regenerative cell therapy that uses myoblasts, or muscle stem cells,that are grown from a patient's own muscle and genetically engineered tooverexpress SDF-1 have demonstrated pre-clinical efficacy for treatingheart failure and are being tested on patients in clinical trials torepair ischemically damaged tissue and increase function in the REGENtrial. Taken together, these published data from multiple laboratoriesdemonstrate that, independent of the delivery method, overexpression ofSDF-1 provides functional benefit in diseases of ischemic etiology.

Re-stimulating SDF-1 expression in ischemic muscle has a hightherapeutic potential for treatment of CLI by regenerating vasculaturedamaged by poor blood flow. This provides an opportunity to repair andretain function in degenerating limbs. Re-growth of blood vesselarchitecture has been shown to improve limb salvage rates in a number ofclinical trials using VEGF or stem cells. Yamaguchi et al. reported thatlocal delivery of SDF-1 protein enhanced neovascularization of anischemic hindlimb after administration of EPCs, suggesting that SDF-1augments EPC-induced vasculogenesis. Similarly, Hiasa et al.demonstrated that SDF-1 gene transfer enhanced ischemia-inducedvasculogenesis and angiogenesis in vivo through a VEGF/eNOS-relatedpathway.

These observations were recently confirmed in analyses of skeletalmuscle acquired from patients undergoing peri-genicular amputation forchronic CLI. Expression of SDF-1 and its receptor, CXCR4, was increasedin skeletal muscle fibers and microvessels, respectively. This suggeststhat the SDF-1/CXCR4 repair axis has been chronically upregulated inischemic leg muscles in an attempt to stimulate microvessel growth fornatural healing, but is insufficient at such low levels. By usingACRX-100 gene therapy to amplify the signal, we may be able tosynergistically build on the healing process already started in thedamaged tissue. Thus, ACRX-100 represents a novel chemokine therapy fornext generation therapeutic neovascularization.

Summary of Master Cell Bank and Bulk Plasmid Production

The manufacturing of drug substance used for the heart failure Phase Iclinical trial was accomplished at a scale of 300 L using dedicatedreagents and materials. For our proposed Phase I/II CLI trials, we willbe able to leverage our experience gained by utilizing our qualifiedMaster Cell Bank and streamline drug product manufacture usingpre-established quality testing services. All media are prepared withcommon animal-free ingredients and sterile USP-grade water or betterquality. All buffers for bacterial lysis are released for manufacturingbased on the respective certificates of analysis. Large-scale lysis isaccomplished via a bubble device, without use of RNase. Allchromatographic buffers are manufactured at Aldevron with USP-grade orequivalent reagents and released after meeting internal specifications.All aseptic processing are performed in environmentally-controlled cleanrooms. The clean room facility consists of an anteroom (Class 10,000,ISO 7) for gowning with two doors and a pass-through window to the mainclean room (Class 10,000); inside is a class 1000 (ISO 6) room with aclass-100 (ISO 5) biosafety hood for final fill/finish. Procedures arein place for use and maintenance, cleaning (after every batch), andEnvironmental Monitoring. The bulk plasmid solution isfilter-sterilized, the concentration adjusted as necessary, and storedat −75+5° C. for final dispensing into drug product vials.

The final step in manufacture is aseptic dilution of sterile solutionsinto sterile bulk containers. Therefore, for early development, the drugsubstance was specified to be sterile.

Summary of Drug Product Manufacture

The bulk plasmid is transferred into a class ISO 6 clean room thathouses an ISO 5 biological-safety cabinet (BSC). All contact materialsare pre-sterilized, and pyrogen free, disposable items that have beenreleased for use based on manufacturer's certificate of analysis.Processing occurs in the BSC. The bulk plasmid solution was firstdiluted to the final target concentration with USP Dextrose (5%) forinjection. After mixing, the sterile plasmid solution is then manuallypipetted into the final pre-sterilized serum vials. Product is storedfrozen at −75+5° C. A comprehensive list of media components anddownstream reagents, along with quality information, will be provided inthe IND.

Stability

Stability studies have demonstrated that pre-clinical lots of ACRX-100are stable at −20° C. for up to a year from manufacture. Clinical gradeACRX-100 (Lots 24370D-F) are currently under accelerated (5±3° C.) andstandard (−20±4° C.) stability (Table 1) and results are expected toexceed preclinical stability findings. Results will be reported in theIND. The following tests will be performed as part of the stabilityprogram: appearance, identity (agarose gel electrophoresis),concentration (A260/A280), homogeneity (densitometry), potency, and pH.Sterility testing will be performed at release, six months, and annuallythereafter.

Stability Conditions and Time Points for Clinical Trial Lots 24370D-FTest Intervals (months) Storage Conditions 0 1 3 6 9 12 18 24 −20° C. ±4° C. XY X X XY X XY X XY 5° C. ± 3° C. X X XY X = All conditions, savesterility Y = Sterility

Non-Clinical Pharmacology and Toxicology of ACRX-100

“Chemokine pharmaceuticals” have recently attracted substantial interestdue to their ability to stimulate and recruit stem cells to sites oftissue injury. ACRX-100 is a gene therapy agent that delivers the humanchemokine SDF-1 via gene expression in human cells. The active proteinproduced by ACRX-100, SDF-1, has been shown to improve cardiac functionin temporally remote ischemically-damaged myocardium and improve thehealing rate of wounded epithelia by recruiting CXCR4-positive stemcells. SDF-1 has shown pro-angiogenic activity in patients with acuteCLI and is down-regulated in patients with chronic CLI, suggesting thattherapies directed at renewing SDF-1 expression in chronic CLI mayaugment vasculogenesis via recruitment of bone-marrow derived cells tothe adult vasculature.

Plasmid ACL-01110Sk, formulated in the drug product ACRX-100, has beentested in animal models of ischemic cardiovascular disease and hind limbischemia. ACRX-100 was tested via intra-cardiac administration in aporcine model of heart failure for efficacy, safety and biodistribution,and a No Observed Adverse Effect Level (NOAEL) of 100 mg wasestablished. The identical formulation of ACRX-100, administered atdoses lower than those used in nonclinical and clinical studies of heartfailure, is currently being evaluated to determine the potentialtherapeutic benefit provided in CLI. A single dose efficacy, toxicologyand biodistribution study with ACRX-100 has been conducted in hindlimbischemic rabbits. This study demonstrated that ACRX-100 has therapeuticpotential for the treatment of critical limb ischemia and thatintramuscular injection of ACRX-100 into the ischemic hindlimbs ofrabbits did not produce any signs of toxicity or histopathologicchanges. Additionally, the ACL-01110Sk plasmid was essentially clearedfrom all organs but the ischemic limb at 60 days post-therapy after asingle dose. A repeat-dose efficacy and safety (toxicology andbiodistribution) study is planned in the rabbit model of hindlimbischemia to support up to 3 doses of ACRX-100 in patients with CLI.

The following examples are for the purpose of illustration only and arenot intended to limit the scope of the claims, which are appendedhereto.

Example 1

Stromal cell-derived factor-1 or SDF-1 is a naturally-occurringchemokine whose expression is rapidly upregulated in response to tissueinjury. SDF-1 induction stimulates a number of protectiveanti-inflammatory pathways, causes the down regulation ofproinflammatory mediators (such as MMP-9 and IL-8), and can protectcells from apoptosis. Furthermore, SDF-1 is a strong chemoattractant oforgan specific and bone marrow derived stem cells and progenitor cellsto the site of tissue damage, which promotes tissue preservation andblood vessel development. Based on observations that increasedexpression of SDF-1 led to improved cardiac function in ischemic animalmodels, we focused on developing a non-viral, naked-DNA SDF-1-encodingplasmid for treatment of ischemic cardiovascular disease. During thecourse of development, the plasmid was optimized based on cell cultureand small animal study results described below. The ACL-01110Sk plasmidwas selected based on its ability to express transgenes in cardiactissue and to consistently improve cardiac function in pre-clinicalanimal models of ischemic cardiomyopathy. SDF-1 transgene expression inACL-01110Sk is driven by the CMV enhancer/promoter, CMV intron A, andthe RU5 translational enhancer. The drug product, JVS-100 (formerlyACRX-100), is composed of the ACL-01110Sk plasmid in 5% dextrose.

Initial studies in a rat model of heart failure demonstrated thatACL-01110S (an SDF-1 expressing precursor to ACL-01110Sk) improvedcardiac function after injection of the plasmid directly into theinfarct border zone of the rat hearts four weeks following an MI.Benefits were sustained for at least 8-10 weeks post-injection andcorrelated with increased vasculogenesis in the ACL-01110S treatedanimals. ACL-01110S was modified to optimize its expression profile. Theplasmid ACL-01110Sk was deposited with the Amer. Type Cult. Coll. (10801University Blvd., Manassas, Va. 20110-2209) on Nov. 14, 2012 and hasbeen assigned Accession No. PTA-13320.

Plasmid Dose-Dependent Expression in a Rat Model of MI

To determine the plasmid dose per injection that would provide maximalexpression in rat cardiac tissue, escalating doses (10, 50, 100, 500 μg)of the ACL-00011L luciferase plasmid were injected into infarcted rathearts. Lewis rats were subjected to a median sternotomy and the leftanterior descending artery (LAD) was permanently ligated, and injectedperi-MI at one site with 100 μl ACL-00011L plasmid in PBS. Whole bodyluciferase expression was measured in each dose cohort (n=3) bynon-invasive bioluminescent imaging (Xenogen, Hopkinton, Mass.) atbaseline and at 1, 2, 3, 4, and 5 days post-injection. The peakexpression increased up to a dose of 100 μg and saturated at higherdoses. Based on this dose-response curve, a dose of 100 μg wasdetermined to be sufficient for maximal plasmid expression in rathearts. ACL-00011L expressed the luciferase gene from a vector backboneequivalent to that used in construction of ACL-00011S, which expressesSDF-1.

Comparison of Cardiac Vector Expression in a Rat Model of Ischemic HeartFailure

The luciferase expressing equivalents of several SDF-1 plasmidcandidates were tested for expression in cardiac tissue in a rat modelof myocardial infarct (MI). Plasmid candidates differed in the promotersdriving expression and presence of enhancer elements. Lewis rats weresubjected to a median sternotomy and the left anterior descending artery(LAD) was permanently ligated and the chest was closed. Four weekslater, the chest was reopened, and the luciferase expressing plasmidswas directly injected (100 μg in 100 μA per injection) into 4peri-Myocardial infarction sites. At 1, 2, 4, 6, 8, and 10 dayspost-injection (and every 3-4 days following), rats were anesthetized,injected with luciferin and imaged with a whole-body Xenogen Luciferaseimaging system.

The two CMV driven plasmids tested, ACL-00011L and ACL-01110L yieldeddetectable luciferase expression within 24 hours of injection with aninitial peak of expression at 2 days post-injection.

ACL-01110L peak expression was 7 times greater than ACL-00011L andexpression was approximately 10 days longer (lasting up to 16 days postinjection). In contrast, ACL-00021L (αMHC driven plasmid) showed noinitial peak, but expressed at a low-level through day 25post-injection. These results support previous studies demonstratingthat CMV driven plasmids can be used for localized, transient proteinexpression in the heart and that the timeframe of therapeutic proteinexpression can be modulated through the inclusion of enhancer elements.

Efficacy of SDF-1 Plasmids in Rat Model of MI

SDF-1-encoding plasmids were tested in a rat model of MI to determine iffunctional cardiac benefit could be achieved. Lewis rats were subjectedto a median sternotomy and the LAD was permanently ligated immediatelydistal to the first bifurcation. Four weeks later, the chest wasreopened, and one of three SDF-1 expressing plasmids (ACL-01110S,ACL-00011S, or ACL-000215) or saline was injected (100 μg per 100 μlinjection) into 4 peri-MI sites.

At baseline (pre-injection), and 2, 4, and 8 weeks post-injection, ratswere anesthetized and imaged with M-mode echocardiography. LVEF,fractional shortening, and LV dimensions were measured by a trainedsonographer who was blinded to randomization.

A strong trend in improvement in cardiac function was observed with bothCMV driven plasmids, ACL-011105 and ACL-00011S, compared to salinecontrols. ACL-011105 elicited a statistically significant increase infractional shortening at four weeks that was sustained 8 weeks afterinjection. In contrast, no difference in function was observed betweenαMHC driven plasmid ACL-00021S and saline. Furthermore, compared tocontrol, the ACL-011105 and the ACL-00011S-treated animals hadsignificant increases in large vessel density (ACL-01110S: 21±1.8vessels/mm²; ACL-00011S: 17±1.5 vessels/mm²; saline: 6±0.7 vessels/mm²,p<±0.001 for both vs. saline) and reduced infarct size (ACL-01110S:16.9±2.8%; ACL-000115: 17.8±2.6%; saline: 23.8±4.5%). Importantly,treatment with ACL-01110S demonstrated the largest improvement incardiac function and vasculogenesis, and caused the largest reduction ininfarct size.

In summary, in a rat model of ischemic heart failure, bothSDF-1-encoding plasmids driven by a CMV promoter provided functionalcardiac benefit, increased vasculogenesis, and reduction in infarct sizecompared to saline treatment. In all parameterstested, ACL-011105provided the most significant benefit.

Transfection Efficiency of ACL-01110Sk and ACL-01010Sk in H9C2 Cells

In vitro transfection of H9C2 myocardial cells without transfectionreagents (i.e.,—naked plasmid DNA was added to cells in culture) wereused to estimate in vivo transfection efficiencies of GFP versions ofthe inventors lead plasmid vectors, ACL-01110Sk and ACL-01010Sk. H9C2cells were cultured in vitro and various amounts of pDNA (0.5 μg, 2.0μg, 4.0 μg, 5.0 μg) were added in 5% dextrose. The GFP vectors wereconstructed from the ACL-01110Sk (ACL-01110G) or ACL-01010Sk(ACL-01010G) backbones. At Day 3 post-transfection, GFP fluorescence wasassessed by FACS to estimate transfection efficiency. The transfectionefficiencies for the ACL-01110G and ACL-01010G vectors in 5% dextroseranged from 1.08-3.01%. At each amount of pDNA tested, both vectors hadsimilar in vitro transfection efficiencies. We conclude that the 1-3%transfection efficiency observed in this study is in line with findingsfrom previous studies demonstrating a similar level of in vivotransfection efficiency. Specifically, JVS-100 will transfect a limitedbut sufficient number of cardiac cells to produce therapeutic amounts ofSDF-1.

Example 2 Expression of Plasmid in Porcine Myocardium

A porcine occlusion/reperfusion MI model of the left anterior descendingartery (LAD) was selected as an appropriate large animal model to testthe efficacy and safety of ACRX-100. In this model, 4 weeks recovery isgiven between MI and treatment to allow time for additional cardiacremodeling and to simulate chronic ischemic heart failure.

Surgical Procedure

Yorkshire pigs were anesthetized and heparinized to an activatedclotting time (ACT) of >300 seconds, and positioned in dorsalrecumbency. To determine the contour of the LV, left ventriculographywas performed in both the Anterior-Posterior and Lateral views.

Delivery of Luciferase Plasmid into Porcine Myocardium

A deflectable guide catheter device was advanced to the left ventricleretrograde across the aortic valve, the guide wire was removed, and anLV endocardial needle injection catheter was entered through the guidecatheter into the LV cavity. Luciferase plasmid was injected at 4 sitesat a given volume and concentration into either the septal or lateralwall of the heart. Five combinations of plasmid concentration (0.5, 2,or 4 mg/ml) and site injection volumes (0.2, 0.5, 1.0 ml) were tested.Plasmid at 0.5 mg/ml was buffered in USP Dextrose, all others werebuffered in USP Phosphate Buffered Saline. For each injection, theneedle was inserted into the endocardium, and the gene solution wasinjected at a rate of 0.8-1.5 ml/minute. Following injection, the needlewas held in place for 15 seconds and then withdrawn. After injectionswere completed, all instrumentation was removed, the incision wasclosed, and the animal was allowed to recover.

Harvesting of Myocardial Tissue

On Day 3 post injection, the animals were submitted to necropsy.Following euthanasia, the heart was removed, weighed, and perfused withLactate Ringers Solution until clear of blood. The LV was opened and theinjection sites identified. A 1 cm square cube of tissue was takenaround each injection site. Four (4) cubes harvested from the posteriorwall remote from any injection sites served as negative controls. Thetissue samples were frozen in liquid nitrogen and stored at −20 to −70°C.

Assessment of Luciferase Expression

The tissue samples were thawed and placed in a 5 ml glass tube. Lysisbuffer (0.5-1.0 ml) was added and tissue was disrupted using Polytronhomogenization (model PT1200) on ice. Tissue homogenate was centrifugedand protein concentration of the supernatant was determined for eachtissue sample using the Bio-rad Detergent-Compatible (DC) protein assayand a standard curve of known amounts of bovine serum albumin (BSA).Tissue sample homogenate (1-10 μl) was assayed using the Luciferaseassay kit (Promega).

The results of the experiment are shown in FIG. 1. The data shows thatexpression of the vector increases with increasing injection volume andincreasing concentration of DNA.

Example 3 Improvement in Cardiac Function by SDF-1 Plasmid Treatment inPorcine Model of Ischemic Cardiomyopathy Induction of MyocardialInfarction

Yorkshire pigs were anesthetized and heparinized to an activatedclotting time (ACT) of ≧250 seconds, and positioned in dorsalrecumbency. A balloon catheter was introduced by advancing it through aguide catheter to the LAD to below the first major bifurcation of theLAD. The balloon was then inflated to a pressure sufficient to ensurecomplete occlusion of the artery, and left inflated in the artery for90-120 minutes. Complete balloon inflation and deflation was verifiedwith fluoroscopy. The balloon was then removed, the incision was closed,and the animal was allowed to recover.

Enrollment Criteria

One month post-MI, cardiac function in each pig was assessed byechocardiography. If the LVEF was less than 40% and the LVESV wasgreater than 56.7 ml, the pig was enrolled in the study.

Surgical Procedure

Each enrolled pig was anesthetized and heparinized to an activatedclotting time (ACT) of ≧300 seconds, and positioned in dorsalrecumbency. To determine the contour of the LV, left ventriculographywas performed in both the Anterior-Posterior and Lateral views.

Delivery of SDF-1 plasmid (ACL-01110Sk) into Myocardium

Each pig was randomized to one of 3 sacrifice points: 3 days, 30 days,or 90 days post-treatment, and to one of four treatment groups: control(20 injections, buffer only), low (15 injections, 0.5 mg/ml), mid (15injections, 2.0 mg/ml), or high (20 injections, 5.0 mg/ml). All plasmidwas buffered in USP Dextrose. The injection procedure is describedbelow.

A deflectable guide catheter device was advanced to the left ventricleretrograde across the aortic valve, the guide wire was removed, and anLV endocardial needle injection catheter was entered through the guidecatheter into the LV cavity. SDF-1 plasmid or buffer at randomized dosewas loaded into 1 ml syringes that were connected to the catheter. Eachinjection volume was 1.0 ml. For each injection, the needle was insertedinto the endocardium, and the solution was injected over 60 seconds.Following injection, the needle was held in place for 15 seconds andthen withdrawn. After injections were completed, all instrumentation wasremoved, the incision was closed, and the animal was allowed to recover.

At sacrifice, samples of tissues from the heart and other major organswere excised and flash frozen for PCR and histopathological analysis.

Assessment of Cardiac Function

Each animal had cardiac function assessed by standard 2-dimensionalechocardiography at day 0, 30, 60, and 90 post-injection (or untilsacrifice). Measurements of left ventricular volume, area, and wallmotion score were made by an independent core laboratory. The efficacyparameters measured are shown below in Table 1.

TABLE 1 Echocardiographic Parameters Variable Name Definition LVESV EndSystolic Volume measured in parasternal long-axis view LVEDV EndDiastolic Volume measured in parasternal long-axis view LVEF(LVEDV-LVESV)/LVEDV *100% WMSI Average of all readable wall motionscores based on ASE 17 segment model and scoring system of 0-5.

The impact of SDF-1 plasmid on functional improvement is shown in FIGS.2-5. FIGS. 2-4 show that the low and mid doses of SDF-1 plasmid improveLVESV, LVEF, and Wall Motion Score Index at 30 days post-injectioncompared to control; whereas, the high dose does not show benefit. FIG.5 demonstrates that the cardiac benefit in the low and mid dose issustained to 90 days, as both show a marked attenuation in pathologicalremodeling, that is, a smaller increase in LVESV, compared to control.

Assessment of Vasculogenesis

Animals that were sacrificed at 30 days were assessed for vessel densityin the leftventricle using 7 to 9 tissue samples harvested from eachformalin-fixed heart. Genomic DNA was extracted and efficiently purifiedfrom formalin-fixed tissue sample using a mini-column purificationprocedure (Qiagen). Samples from SDF-1 treated and control animals weretested for presence of plasmid DNA by quantitative PCR. Three to fivetissue samples found to contain copies of plasmid DNA at least 4-foldabove background (except in control animals) for each animal were usedto prepare slides and immunostained with isolectin. Cross-sections wereidentified and vessels counted in 20-40 random fields per tissue. Thevessels per field were converted to vessels/mm² and were averaged foreach animal. For each dose, data is reported as the average vessels/mm²from all animals receiving that dose.

FIG. 6 shows that both doses that provided functional benefit alsosignificantly increase vessel density at 30 days compared to control. Incontrast, the high dose, which did not improve function, did notsubstantially increase vessel density. This data provides a putativebiologic mechanism by which SDF-1 plasmid is improving cardiac functionin ischemic cardiomyopathy.

Biodistribution Data

JVS-100 distribution in cardiac and non-cardiac tissues was measured 3,30 and 90 days after injection in the pivotal efficacy and toxicologystudy in the pig model of MI. In cardiac tissue, at each time point,average JVS-100 plasmid concentration increased with dose. At each dose,JVS-100 clearance was observed at 3, 30 and 90 days following injectionwith approximately 99.999999% cleared from cardiac tissue at Day 90.JVS-100 was distributed to non-cardiac organs with relatively high bloodflow (e.g. heart, kidney, liver, and lung) with the highestconcentrations noted 3 days following injection. JVS-100 was presentprimarily in the kidney, consistent with renal clearance of the plasmid.There were low levels of persistence at 30 days and JVS-100 wasessentially undetectable in non-cardiac tissues at 90 days.

Conclusions

Treatment with JVS-100 resulted in significantly increased blood vesselformation and improved heart function in pigs with ischemic heartfailure following a single endomyocardial injection of 7.5 and 30 mg.The highest dose of JVS-100 tested (100 mg) showed a trend in increasedblood vessel formation but did not show improved heart function. None ofthe doses of JVS-100 were associated with signs of toxicity, adverseeffects on clinical pathology parameters or histopathology. JVS-100 wasdistributed primarily to the heart with approximately 99.999999% clearedfrom cardiac tissue at 90 days following treatment. JVS-100 wasdistributed to non-cardiac organs with relatively high blood flow (e.g.,heart, kidney, liver, and lung) with the highest concentrations in thekidneys 3 days following injection. JVS-100 was essentially undetectablein the body 90 days after injection with only negligible amounts of theadministered dose found in non-cardiac tissues. Based on these findingsthe no observed adverse effect level (NOAEL) for JVS-100 in the pigmodel of MI was 100 mg administered by endomyocardial injection.

Example 4 Porcine Exploratory Study: LUC Injections by TransarterialInjection in Chronic MI Pigs Methods

One pig with a previous LAD occlusion/reperfusion MI and an EF>40%, wasinjected with ACL-01110Sk with a Transarterial catheter. Two injectionsin the LAD and 2 in the LCX were performed with an injection volume of2.5 ml and a total injection time of 125-130 sec. One additionalinjection in the LCX of 3.0 ml with a total injection time of 150 secwas performed with contrast mixed with the plasmid.

Sacrifice and Tissue Collection

Three days following the injections, the animal was euthanized. Aftereuthanasia, the heart was removed, drained of blood, placed on an icecold cutting board and further dissected by the necropsy technician orpathologist. The non-injected myocardium from the septum was obtainedvia opening the right ventricle. The right ventricle was trimmed fromthe heart and placed in cold cardioplegia. New scalpel blades were usedfor each of the sections.

Next, the left ventricle was opened and the entire left ventricle wasexcised by slicing into 6 sections cutting from apex to base. The LV wasevenly divided into 3 slices. Following excision, each section was ableto lay flat. Each section (3 LV sections, 1 RV section, and 1 pectoralmuscle) was placed in separate labeled containers with cold cardioplegiaon wet ice, and transported for luciferase analysis.

Luciferase Imaging

All collected tissues were immersed in luciferin and imaged with aXenogen imaging system to determine plasmid expression.

Results

A representative image of the heart is shown in FIG. 8. The coloredspots denote areas of luciferase expression. These spots showed RelativeLight Units (RLUs) of greater than 10⁶ units, more than 2 orders ofmagnitude above background. This data demonstrated that the catheterdelivered plasmid sufficient to generate substantial plasmid expressionover a significant portion of the heart.

Example 5 Clinical Study Example

Ascending doses of JVS-100 are administered to treat HF in subjects withischemic cardiomyopathy. Safety is tracked at each dose by documentingall adverse events (AEs), with the primary safety endpoint being thenumber of major cardiac AEs at 30 days. In each cohort, subjects willreceive a single dose of JVS-100. In all cohorts, therapy efficacy isevaluated by measuring the impact on cardiac function via standardechocardiography measurements, cardiac perfusion via Single PhotonEmission Computed Tomography (SPECT) imaging, New York Heart Association(NYHA) class, six minute walk distance, and quality of life.

All subjects have a known history of systolic dysfunction, prior MI, andno current cancer verified by up to date age appropriate cancerscreening. All subjects are screened with a physician visit, and acardiac echocardiogram. Further baseline testing such as SPECT perfusionimaging, is performed. Each subject receives fifteen (15) 1 mlinjections of JVS-100 delivered by an endocardial needle catheter tosites within the infarct border zone. Three cohorts (A, B, C) will bestudied. As shown in Table 2, dose will be escalated by increasing theamount of DNA per injection site while holding number of injection sitesconstant at 15 and injection volume at 1 ml. Subjects are monitored forapproximately 18 hours post-injection and have scheduled visits at 3 and7 days post-injection to ensure that there are no safety concerns. Thepatient remains in the hospital for 18 hours after the injection toensure all required blood collections (i.e., cardiac enzymes, plasmaSDF-1 protein levels) are performed. All subjects have follow-up at 30days (1 month), 120 days (4 months), and 360 days (12 months) to assesssafety and cardiac function. The primary safety endpoint are majoradverse cardiac events (MACE) within 1 month post-therapy delivery. AEswill be tracked for each subject throughout the study. The followingsafety and efficacy endpoints will be measured:

Safety:

Number of Major Adverse Cardiac Events (MACE) at 30 days post-injection

Adverse Events throughout the 12 month follow-up period

Blood lab Analysis (Cardiac Enzymes, CBC, ANA)

SDF-1 Plasma Levels

Physical assessment

Echocardiography

AICD monitoring

ECG

Efficacy:

Change from baseline in LVESV, LVEDV, LVEF, and wall motion score index

Change from baseline in NYHA classification and quality of life

Change from baseline in perfusion as determined by SPECT imaging

Change from baseline in Six Minute Walk Test distance

TABLE 2 Clinical Dosing Schedule Amount # Total # of of InjectionInjection Dose Cohort Subjects DNA/site volume/site Sites of DNA CohortA 4 0.33 mg  1.0 ml 15  5 mg Cohort B 6 1.0 mg 1.0 ml 15 15 mg Cohort C6 2.0 mg 1.0 ml 15 30 mg

Based on preclinical data, delivery of JVS-100 is expected to elicit animprovement of cardiac function and symptoms at 4 months that sustainsto 12 months. At 4 months following JVS-100 injection, compared tobaseline values, an improvement in six minute walk distance of aboutgreater than 30 meters, an improvement in quality of life score of about10%, and/or an improvement of approximately 1 NYHA class areanticipated. Similarly, we expect a relative improvement in LVESV, LVEF,and/or WMSI of approximately 10% compared to baseline values.

Example 6 Evaluation of Cardiac Function by Echocardiography in ChronicHeart Failure Pigs after Treatment with ACL-01110Sk or ACL-01010SkPurpose

The purpose of this study is to compare functional cardiac response toACL-01110Sk or ACL-01010Sk after endomyocardial catheter delivery in aporcine model of ischemic heart failure.

This study compared efficacy of ACL-01110Sk and ACL-01010Sk in improvingfunction in a porcine ischemic heart failure model. In this study, theplasmids were delivered by an endoventricular needle injection catheter.Efficacy was assessed by measuring the impact of the therapy on cardiacremodeling (i.e., left ventricular volumes) and function (i.e., leftventricular ejection fraction (LVEF)) via echocardiography.

Methods

Briefly, male Yorkshire pigs were given myocardial infarctions by LADocclusion via balloon angioplasty for 90 minutes. Pigs having anejection fraction <40% as measured by M-mode echocardiography 30 dayspost-infarct were enrolled. Pigs were randomized to one of 3 groups tobe injected with either Phosphate Buffered Saline (PBS, control),ACL-01110Sk or ACL-01010Sk in PBS using an endoventricular needleinjection catheter delivery system (Table 3).

TABLE 3 Initial Study Design: SDF-1 Therapy for Chronic Heart Failure inPigs Injection Amount of #Injection Group Plasmid #of Pigs volume/siteDNA/site Sites Total DNA 1 Vehicle 3 200 μl N/A 10 n/a 2 ACL-01010Sk 3200 μl 400 μg 10 4 mg 3 ACL-01110Sk 3 200 μl 400 μg 10 4 mg

Echocardiograms were recorded prior to injection and at 30 and 60 dayspost-injection. Table 4 below defines the variables as they are referredto in this report.

TABLE 4 Definition of variables Variable Name Definition LVESV EndSystolic Volume measured in parastemal long-axis view LVEDV EndDiastolic Volume measured in parastemal long-axis view LVEF(LVEDV-LVESV)/LVEDV *100%

Results

The baseline echocardiographic characteristics at time of initialinjection (Day 30 post-MI) for all enrolled animals in this report (n=9)as reported by the echocardiography core laboratory, are provided inTable 5 below.

TABLE 5 Baseline characteristics Baseline Value Baseline Value BaselineValue Parameter Group 1 Group 2 Group 3 LVESV  78 ± 18 ml 67 ± 2 ml 86 ±31 ml LVEDV 132 ± 30 ml 114 ± 11 ml 130 ± 36 ml  LVEF 41 ± 1%  41 ± 5% 34 ± 10% 

Table 5 shows the LVESV, LVEF and LVEDV at 0 and 30 days post-initialinjection. Control PBS animals demonstrated an increase in LVESV andLVEDV and no improvement in LVEF consistent with this heart failuremodel. The treatment groups did not reduce cardiac volumes or increaseLVEF compared to control. Similar results were obtained at 60 dayspost-initial injection.

Example 7

A strategy to augment stem cell homing to the peri-infarct region bycatheter-based transendocardial delivery of SDF-1 in a porcine model ofmyocardial infarction was investigated to determine if it would improveleft ventricular perfusion and function. The catheter-based approach hasbeen used successfully for cell transplantation and delivery ofangiogenic growth factors in humans.

Female German landrace pigs (30 kg) were used. After an overnight fast,animals were anesthetized and intubated.

A 7 French sheath was placed in the femoral artery with the animal in asupine position. An over-the-wire balloon was advanced to the distalLAD. The balloon was inflated with 2 atm and agarose beads were injectedslowly over 1 min via the balloon catheter into the distal LAD. After 1minute the balloon was deflated and the occlusion of the distal LAD wasdocumented by angiography. After induction of myocardial infarctionanimals were monitored for 3-4 h until rhythm and blood pressure wasstable. The arterial sheath was removed, carprofen (4 mg/kg) wasadministered intramuscularly and the animals were weaned from therespirator. Two weeks after myocardial infarction animals wereanesthetized. Electromechanical mapping of the left ventricle wasperformed via an 8F femoral sheath with the animal in the supineposition. After a complete map of the left ventricle had been obtained,human SDF-1 (Peprotec, Rocky-Hill, N.J.) was delivered by 18 injections(5 μg in 100 ml saline) into the infarct and periinfarct region via aninjection catheter. 5 μg per injection were used to adjust for thereported efficiency of the catheter injection. Injections were performedslowly over 20 s and only when the catheter's tip was perpendicular tothe left ventricular wall, when loop stability was <2 mm and when needleprotrusion into the myocardium provoked ectopic ventricular extra beats.Control animals underwent an identical procedure with sham injections.Echocardiography excluded postinterventional pericardial effusion.

Twenty (20) animals completed the study protocol: 8 control animals and12 SDF-1 treated animals. For myocardial perfusion imaging only 6control animals could beevaluated due to technical problems. Infarctlocation was anteroseptal in all animals.

Infarct size in percent of the left ventricle as determined bytetrazolium staining was 8.9±2.6% in the control group and 8.9±1.2% inthe SDF-1 group. Left ventricular muscle volume was similar in bothgroups (83±14 ml versus 95±10 ml, p=ns). Immunofluorescence stainingrevealed significantly more vWF-positive vessels in the peri-infarctareain SDF-1 treated animals than in control animals (349±17/mm² vs.276±21/mm², p<0.05). A profound loss of collagen in the peri-infarctarea was observed in SDF-1 treated animals as compared to controlanimals (32±5% vs. 61±6%, p<0.005). The number of inflammatory cells(neutrophils and macrophages) within the peri-infarct area was similarin both groups (332±51/mm² vs. 303±55/mm², p=ns). Global myocardialperfusion did not change from baseline to follow-up SPECT and there wasno difference between groups. Final infarct size was similar in bothgroups and compared well to the results of tetrazolium stainingSegmental analysis of myocardial perfusion revealed decreased traceruptake in apical and anteroseptal segments with significant differencesbetween myocardial segments. However, tracer uptake at baseline andfollow-up were nearly identical in control and SDF-1 treated animals.There were no differences in end diastolic and end systolic volumesbetween groups. However, stroke volume increased in control animals anddecreased slightly in SDF-1 treated animals. The difference between bothgroups was significant.

Similarly, ejection fraction increased in control animals and decreasedin SDF-1 treated animals. The difference between groups showed a strongtrend (p=0.05). Local shortening, another parameter of ventricularmechanical function, did not change in control animals. However, localshortening decreased significantly in SDF-1 treated animals, resultingin a significant difference between groups. There were no significantdifferences in unipolar voltage within and between groups. Significantcorrelations between baseline ejection fraction and stroke volume andbaseline local shortening (EF and LS: r=0.71, SV and LS: r=0.59) werenoted. Similar results were obtained for follow-up values (EF and LS:r=0.49, SV and LS: r=0.46). The change in local shortening correlatedsignificantly with the change in ejection fraction (r=0.52) and strokevolume (r=0.46). There was neither a correlation between localshortening and end diastolic volume (baseline r=−0.03, follow-up r=0.12)nor between ejection fraction and end diastolic volume (baseliner=−0.04, follow-up r=0.05). Segmental analysis of EEM data showeddecreased unipolar voltage and local shortening in the anteroseptalsegments with significant differences between myocardial segments atbaseline. The distribution of unipolar voltage values in myocardialsegments was similar in both groups at baseline and at follow-up.Segmental local shortening did not change in the control group. However,it decreased in the SDF-1 group, mainly due to a decrease in the lateraland posterior segment of the left ventricle. There was a significantinteraction between assignment to SDF-1 and follow-up vs. baseline.

The study described above demonstrated that a single application ofSDF-1 protein was insufficient to produce functional cardiac benefit.

Example 8 ACRX-100 Vector Time-Course Expression in a Rat Model ofHindlimb Ischemia Purpose

The purpose of this study was to establish the duration of ACRX-100vector expression after direct intramuscular injection in a rodent modelof hindlimb ischemia.

Methods

ACRX-100 Lot #25637 was manufactured by Aldevron, LLC (Fargo, N. Dak.).Male Lewis rats were anesthetized and a longitudinal incision in themedial thigh from the inguinal ligament to the knee joint, exposing thefemoral artery, which was ligated and removed. Animals were allowed torecover for 10 days, then anesthetized and directly injected with 1.0,2.0 or 4 mg/ml of ACL-01110L (vector backbone with luciferase cDNA) in0.2 ml at 4 sites along the hindlimb. Vector expression was routinelymeasured for luciferase expression at days 1, 2, 3, 8, 10, and 14 usinga cooled couple device camera from Xenogen Imaging Systems. The animalswere anesthetized using 2% isofluorane and luciferin was injectedintraperitoneally at a concentration of 125 mg/kg of the animal. After10 minutes, real time images were obtained during a 1 minute exposure todetermine the whole body chemiluminescence of luciferase expression.Data was measured as total flux (pixels/second).

Results

Similar to previous studies, CMV driven plasmid ACL-01110L had a peakexpression on Day 3 (FIG. 11). Minimal expression was seen after day 14.

Conclusions

ACRX-100 expression in ischemic rat hindlimbs peaked at day 3, and wasexpressed for up to 14 days, consistent with expression patternsmeasured in rat cardiac tissue and previously published studies ofvector expression driven by the CMV-promoter. This data suggests thatfor future studies evaluating efficacy of repeat doses of ACRX-100, a 2week interval between dosing is reasonable. This dosing interval alsocorrelates with dosing regimens reported in several clinical trialsusing CMV-based vectors driving therapeutic gene expression (FGF, HGF,VEGF, HIF1) using naked plasmid DNA to treat ischemic diseases.

Example 9 In Vivo Characterization of ACRX-100 Dosing in Rabbit HindlimbPurpose

The purpose of this study was to determine the effects of injectionvolume, pDNA concentration, and formulation on ACRX-100 pDNA expression3 days after direct injection into rabbit hindlimb muscle.

Methods

Male New Zealand white rabbits of 3.0-4.0 kg, were anesthetized andinjected with luciferase plasmid ACL-01110L. An incision was made in themedial thigh from the inguinal ligament to the knee joint to expose thefemoral artery. Four or eight injections of 0.5-1.0 ml plasmidACL-01110L in 5% dextrose were injected at 0.1 ml per second into theadductor (2 injections), gracilis (1 injection) and semitendinous (1injection) muscles in each rabbit leg. Injections were categorized into6 groups according to FIG. 13. Each injection site was identified with anylon suture for future identification and the wound was suture closed.Each hindlimb was wrapped with a compression bandage for approximately15 minutes. Three days post-injection, animals were sacrificed and thehindlimb muscles comprising the injection site were removed, soaked inluciferin (15 mg/ml) for 7 minutes and bioluminescence imaged using theIVIS Xenogen machine (FIG. 12). Total flux (pixels per second) wasassessed after a 1 minute exposure.

Results

Macroscopic evaluation of injection sites revealed no inflammation andplasmid DNA was well-tolerated in all animals at all doses. As shown inFIG. 13, expression was observed at all doses delivered, with expressionincreasing as a function of pDNA concentration. Expression appeared toplateau at a concentration of 2 mg/ml and a total DNA dose of 4 mg(Groups 4-6). Higher volume or number of injection sites did notincrease expression, suggesting that pDNA concentration is an importantfactor in adequate muscle cell transfection.

Conclusions

This study demonstrated that a luciferase version of the ACRX-100 vectoris capable of expressing gene product in rabbit hindlimb muscles insufficient quantities for detection 3 days post-injection. Furthermore,this study demonstrated that naked plasmid DNA expression in rabbit hindlimb increases with pDNA concentration, up to 2 mg/ml. This studysuggests that 4-8 injection sites, using 0.5-1.0 ml per injection at0.5-2.0 mg/ml pDNA should produce SDF-1 in the rabbit hindlimb.

Example 10 Completed Efficacy, Safety and Biodistribution Studies ofACRX-100

The efficacy, safety and biodistribution of ACRX-100 were previouslydetermined in a GLP Porcine Efficacy, Toxicity and Biodistribution Studyafter direct catheter-mediated cardiac injection in a pig heart failuremodel (summarized in Example 3). ACRX-100 demonstrated an acceptablesafety profile at doses up to 100 mg after direct injection intoischemic pig hearts. This study is considered supportive of the plannedclinical studies in CLI but does not mimic the specific clinicalindication being studied.

The definitive nonclinical assessment of the efficacy, safety andbiodistribution of ACRX-100 supporting the Phase 1 clinical trial in CLIpatients is a rabbit model of hindlimb ischemia. This model provides anexperimental setting that simulates the proposed Phase 1 clinical trialin CLI patients. A safety and efficacy study examining the toxicologyand biodistribution of escalating singles doses of ACRX-100 wasconducted in a rabbit HLI model and is summarized below. Based on theseresults, we are proposing to assess the repeat dose efficacy, toxicologyand biodistribution of ACRX-100 in the rabbit model, outlined in section6.3 of the Pre-IND submission.

Single Dose Safety and Efficacy of ACRX-100 in Rabbit HLI Purpose

The purpose of this study is to evaluate the efficacy, safety andbiodistribution after a single dose of the test article, ACRX-100, in arabbit model of hind limb ischemia.

Methods

New Zealand white rabbits (n=5/group; 2-3 males/females per group)underwent a unilateral femoral artery ligation and 10-days post ligationreceived 4, 8 or 16 mg ACRX-100 or 4 mg of control (luciferase) plasmidvia 8 direct intramuscular 0.5 ml injections to the ischemic limb (Table6).

TABLE 6 Study Design of Safety and Efficacy of ACRX-100 in a rabbitmodel of HLI Table 6. Study design of Safety and Efficacy of ACRX-100 ina rabbit model of HLI # Safety # # pDNA/ Inj. Volume/ Cone pDNA/ RepeatAssessment (Day Group Animals Doses Dose Sites Site (mg/ml) site DoseEfficacy 60) 1 5 1 Control 8 0.5 ml 1 0.5 mg No 0, 30, Biodist/Histopath(4 mg 60 pLuc) 2 5 1 4 mg 8 0.5 ml 1 0.5 mg No 0, 30, Histopath 60 3 5 18 mg 8 0.5 ml 2 1.0 mg No 0, 30, Histopath 60 4 5 1 16 mg  8 0.5 ml 42.0 mg No 0, 30, Biodist/ 60 Histopath

Safety endpoints were evaluated at 60 days post-injection and includedhistopathology and biodistribution from the Hind limb (Injection sites),Opposing Hind limb, Heart, Lung, Liver, Brain, Spleen, Lymph nodes,Kidney, and Ovaries. Gross and microscopic examination of fixedhematoxylin and eosin-stained paraffin sections was performed onsections of tissues as indicated. Clinical pathology was assessed in allgroups at 60 days post-injection. Efficacy was measured by % change inangiographic score compared to control at 30 and 60 days post-treatment.Gastrocnemius muscles were excised and assessed for weight differences60 days post-injection. Biodistribution was assessed in the Lung, Liver,Spleen, Lymph Node, Kidney, Brain, Testes, and Ovaries from animals inGroups 1 and 4.

Results Angiogram Analysis

Angiograms were obtained on Day 0 (pre-injection), 30 (±2), and 60 (±2)days post-injection and recorded in a digital format (FIG. 14).Quantitative angiographic analysis of collateral vessel development inthe ischemic limb was performed with a grid overlay composed of 5 mmdiameter squares arranged in rows. The total number of gridintersections in the medial thigh area, as well as the total number ofintersections crossed by a contrast opacified artery, was counted in asingle blinded fashion. An angiographic score was calculated for eachfilm as the ratio of grid intersections crossed by opacified arteriesdivided by the total number of grid intersections in the medial thigh.

Efficacy

The results of the angiographic data found all animals to be similar onthe day of dosing. In the control animals there was a trend toward adecrease in vascular density over the 60 day post dose period. ACRX-100treated animals showed improved blood flow at both time points in the 1mg/mL (Group 2) and 2 mg/mL (Group 3) groups compared to vector injectedcontrol animals, which showed decreased blood flow. Importantly,improvement in the low and mid dose groups was observed at 30 days withsignificant (p<0.05) benefit in the mid dose group sustained at 60 days(FIG. 15). Improvement was also observed in the Group 4 (4 mg/ml)animals 60 days post-injection. A similar trend towards increasedvasculogenesis was observed in the 5 mg/ml animals in the porcine heartfailure study.

Pathology Mortality

All animals survived to the scheduled necropsy with the exception of aGroup 1 animal (505). Animal 505 died during the 30 day follow upangiogram. The animal was submitted to necropsy. The cause of death wasconsidered to be anesthetic related and not a result of test articleadministration.

Animal Observations

Clinical findings were limited to observation of scabbed areas andsparse hair in many of the animals. These findings are commonobservations in animals undergoing this procedure. In addition,inappetence, decreased activity, and few or absent feces was noted inone Group 2 animal (510) and self mutilation of the hindlimb was notedin two Group 4 animals (519 and 520). The observation of self mutilationin animals 519 and 520 was most likely a result of a loss of sensationfrom the injury in the hind limbs of these animals.

Over the course of the study, the animals initially lost weight duringthe first 3-4 weeks post injury. By the end of the study, animals werereturning to baseline bodyweights. The body weight loss is considered tobe a result of the multiple surgical procedures.

Macroscopic

There were no test article-related macroscopic findings in either sex.The few macroscopic observations were considered incidental andunrelated to treatment.

Microscopic

Several sections of skeletal muscle were examined from injections sitesof the hind limb normal region, hindlimb ischemic region, andnon-injection site opposing limb. Tissue sections were examined usingHemotoxylin and Eosin and Masson's Trichrome staining The tissuessections collected from the ischemic region variably and inconsistentlycontained areas of ischemia. The areas of ischemia were characterizedprimarily by minimal to moderate fibrosis and minimal to mild newcapillary formation (neovascularization) and to a lesser extentsubacute/chronic inflammation, hemorrhage, and myofiberdegeneration/necrosis and/or myofiber regeneration. Often the areas offibrosis and neovascularization followed along fascial planes of themuscle. Occasionally foreign material (suture material) was observed andwas often surrounded by a minimal granulomatous inflammatory response.Rare myofiber mineralization was also observed.

There were no test article-related microscopic findings in the remainingtissues examined microscopically (brain, heart, kidney, liver, lung,ovaries, and spleen). The few remaining microscopic observations wereeither, common background findings in rabbits, or incidental, and weretherefore considered unrelated to treatment.

Hematology and Coagulation

There were no biologically relevant differences among hematology andcoagulation parameters between any of the treatment groups in either sexat 60 days post-injection.

Clinical Chemistry

Phosphorus was sequentially decreased in the ACRX-100 Groups 2, 3, and 4relative to the LUC Plasmid control Group 1 in both males (6 to 36%) andfemales (7 to 30%). There were also mild increases in calcium in theACRX-100 Group 3 and 4 relative to the LUC Plasmid control group in bothmales (4 to 7%) and females (7 to 9%). All mean and individual valuesfor both calcium and phosphorus always remained within expectedhistorical controls ranges. None of these changes were consideredadverse. To better evaluate the veracity of these observations we willassess these values in the repeat dosing safety and efficacy studydescribed in section 6.3. No other alterations among chemistryparameters were observed in either sex.

Biodistribution

In this rabbit CLI study, biodistribution was assessed by quantitativePCR at 60 days post-injection in Group 1 (1 mg/mL luciferase plasmid)and Group 4 (4 mg/mL ACRX-100) animals. The results are shown in Table 7below. As expected, no ACRX-100 vector was detected in tissue from anycontrol group. In the high dose group, ACRX-100 was detected almostexclusively at the injection sites, with only trace amounts of ACRX-100were detected in non-injected organs. The clearance rate of ACRX-100plasmid DNA observed in rabbit hindlimb 60 days post-injection (Table 7)is consistent with the clearance of ACRX-100 from injected cardiac sitesobserved in the porcine heart failure safety study (Table 3, FIG. 16).Additionally, persistence levels were lower in the rabbit CLI study, asthe highest copy number detected in this study at 60 days post-injection(133,360 copies/μg host DNA) was less than the highest copy numberdetected in the cardiovascular study at 90 days (>1×10⁶ copies/μg hostDNA). This data suggests ACRX-100 is being cleared from both injectedand non-injected organs in the rabbit similar to what was demonstratedin the porcine heart failure study, in fact, persistence in the injectedsite in the current rabbit study is less than what was observed in theporcine heart failure model.

TABLE 7 Biodistribution of ACRX-100 in HLI rabbit tissues 60 dayspost-injection Copies of SDF-1 plasmid DNA detected per microgram of DNAfrom tissue or in DNA from equivalent of 10 μL of blood Injection sitenormal tissue Injection site ischemic region Group Sex Animal# BrainHeart 1 2 3 1 2 3 4 1 Male 501 LLOD 23 LLOD LLOD Male 502 LLOD LLOD LLODLLOD Female 503 LLOD LLOD LLOD LLOD Female 504 LLOD LLOD LLOD LLODFemale 505 LLOD LLOD LLOD LLOD 4 Male 516 LLOD LLOD 1106 41 LLOD 11241516 133360 410 Male 517 LLOD LLOD LLOD LLOD LLOD 190 4383 520 3491Female 518 143 LLOD LLOD LLOD 223 LLOD 25974 67455 2616 Female 519 LLODLLOD LLOD LLOD LLOD 391 23 61908 1514 Female 520 LLOD LLOD  32 21 LLOD54744  35682 16910 210 Non-injection site Group Sex Animal# 1 2 KidneyLiver Lung Lymph Ovaries Spleen Blood 1 Male 501 LLOD LLOD Male 502 LLODLLOD Female 503 LLOD LLOD Female 504 LLOD LLOD Female 505 LLOD LLOD 4Male 516 LLOD LLOD LLOD LLOD LLOD LLOD LLOD LLOD LLOD Male 517 LLOD LLODLLOD LLOD LLOD LLOD LLOD LLOD LLOD Female 518 LLOD LLOD LLOD LLOD LLODLLOD LLOD LLOD LLOD Female 519 LLOD LLOD 199 LLOD LLOD LLOD LLOD LLODLLOD Female 520 22 LLOD LLOD LLOD LLOD LLOD LLOD LLOD LLOD LLOD = Lowerthan Limit of Detection.

Conclusions

All animals groups injected with ACRX-100 demonstrated an increase inischemic hindlimb blood flow 60 days post-injection. Animals injectedwith a single intramuscular dose of 1 mg/mL (low dose) or 2 mg/mL (middose) ACRX-100 showed improved blood flow at 30 and 60 days afterinjection compared to vector injected control animals, which showeddecreased blood flow. Importantly, benefits were observed at 30 dayswith significant (p<0.05) benefits sustained at 60 days. These data areconsistent with results from the porcine heart failure studies whereACRX-100 injections (0.5-5.0 mg/mL) resulted in increasedvasculogenesis. Our data suggest that, independent of total ACRX-100delivered to the target tissue, the concentration of product deliveredper injection significantly influences vasculogenesis response. Theseresults indicate that patients with CLI may benefit from a single doseof ACRX-100.

Our data suggests ACRX-100 is being cleared from both injected andnon-injected organs in the rabbit similar to what was demonstrated inthe porcine heart failure study. Since the clearance of ACRX-100 wasconsistent with the cardiovascular study, we submit that the remainingbiodistribution findings of the porcine heart failure safety study holdfor the CLI study as well. Namely, any expression of ACRX-100 at the endof study is minimal, sub-therapeutic and the potential for integrationis less than the spontaneous mutation rate.

Example 11

Proposed Repeat Dose Safety and Efficacy Study of ACRX-100 in RabbitModel of Hindlimb Ischemia

The proposed new study will evaluate the safety and efficacy of repeatdoses of ACRX-100 in the same rabbit model of hindlimb ischemiadescribed in example 10. As discussed below, 3 intramuscular doses ofACRX-100 will be administered to support up to 3 treatments in patientswith CLI.

Safety and Efficacy of ACRX-100 Repeat Dosing in Rabbit HLI Purpose

The purpose of this study will be to determine the safety and efficacyof repeat dosing of ACRX-100 in a rabbit model of hindlimb ischemia.

Rationale

All non-viral gene therapies currently under clinical investigation forcritical limb ischemia deliver their non-viral therapies in repeat doses(HGF, PDGF), including NV1FGF which has demonstrated efficacy in a PhaseII study. To date, our preclinical safety and efficacy studies havedemonstrated that ACRX-100 is safe up to 100 mg after a single cardiacdosing (20 injection sites) or 16 mg hindlimb dosing (8 injectionsites). Therefore, we propose to deliver ACRX-100 in repeat doses at 0,2 and 4 weeks as part of our Phase 1 trial in CLI patients. Repeatdosing of ACRX-100 could provide additional benefit compared to a singledose. CXCR4, the receptor for SDF-1, is upregulated indefinitelyfollowing injury; whereas, SDF-1 is upregulated only transiently afteran acute ischemic event (FIG. 17) or in response to an injectionprocedure. Delivering SDF-1 at multiple later time points followinginjury capitalizes on increased localized expression of CXCR4 expressionin injured tissue and increases stem cell homing to the site of SDF-1expression. In CLI, repeat injections have the potential tosynergistically increase vasculogenesis, collateral vessel growth andwound healing in the ischemic limb. The safety profile observed after100 mg injection into a pig heart suggests that dosing regimens of loweramounts will share similar safety results.

To assess the preclinical safety and efficacy of ACRX-100 repeat dosingin the same model of rabbit hindlimb ischemia used in the study above,we propose the protocol detailed below. The repeat dosing schedule willbe identical to that of the proposed repeat dosing cohorts in the PhaseI/II trial in all aspects except injection volume (described in Example12). In the rabbit, 0.5 mL will be used instead of 1 mL in the human dueto the larger area of skeletal muscle in the human thigh/calf comparedto the rabbit hind limb.

Methods

New Zealand white rabbits (n=5/group of males and females) will undergounilateral femoral artery ligation (day −10) identical to what wasperformed in Example 10, above. Ten days post-ligation (Day 0), eachanimal will receive 3 doses (15 days apart) of 1 mg/mL of controlluciferase plasmid (Groups 1 and 2) or ACRX-100 (Groups 3 and 4)delivered via 16 direct intramuscular injections to the ischemic limb of0.5 mL each (Table 8). Safety endpoints will be evaluated at 3 (day 33)and 30 days (day 60) post-final injection, including histopathology(gross and normal) and biodistribution from the Hind limb (Injectionsites), Opposing Hind limb, Heart, Lung, Liver, Brain, Spleen, Lymphnodes, Kidney, and Ovaries. Gross and microscopic examination of fixedhematoxylin and eosin-stained paraffin sections will be performed onsections of tissues. Clinical chemistry and pathology will be assessedfor all groups prior to hindlimb ischemia (day −10), prior to eachdosing (day 0, 15, 30), 3 days post dosing (day 3, 18, 33), 7 dayspost-final dosing (day 37), and at 30 days post-final dosing (day 60) oruntil sacrifice. Cageside observations will occur twice daily, anddetailed clinical examinations will occur weekly. Efficacy will bemeasured by % change in angiographic score from baseline compared tocontrol at 30 (all cohorts) and 60 (Cohorts 2 and 4) days post-firstinjection.

Samples for biodistribution will be collected from the Lung, Liver,Spleen, Lymph Node, Kidney, Brain, Testes, and Ovaries, and assessed viaqPCR at two sacrifice points: 3 days post-final dosing (day 33) and 30days post-final dosing (day 60).

The 60 day duration post-first treatment of ACRX-100 in the study waschosen to align the efficacy time points with the single treatment studyreported in Example 10. Because this time point is 30 days post-finaldosing, the biodistribution results are expected to have copies ofACRX-100 present at the injection sites (on the order of 10³-10⁵copies/μg host DNA) and limited numbers (<10³ copies/μg host DNA) basedon findings from the heart failure porcine safety and efficacy studyreported in Example 8. The results of this proposed study will bereported in the IND. Assuming that the safety results of this study aresimilar to what was found in the porcine heart failure safety andefficacy study, the study above should justify repeat dosing of ACRX-100in patients with CLI.

TABLE 8 Proposed Repeat Dose Study Design Table 8. Proposed Repeat DoseStudy Design # # pDNA/ #Sites/ Volume/ pDNA/ Treatment Sacrifice Cohortanimals doses Dose Dose site Conc. site times Point Efficacy 1 4 3 8 mg16 0.5 ml 1 mg/ml 0.5 mg Day 0, 15, Day 33 angiograph Control (2 male,30 at day 0, 2 day 30 female) 2 4 Day 60 angiograph Control (2 male, atday 0, 2 day 30, day female) 60 3 5 Day 33 angiograph ACRX- (2 male, atday 0, 100 3 day 30 female) 4 5 Day 60 angiograph ACRX- (3 male, at day0, 100 2 day 30, day female) 60 Safety Assessments in ALL Cohorts:Ante-mortem: Clinical observations, pathology, chemistry Post-mortem:Histopathology and Biodistribution

Example 12 Proposed Clinical Study

The inventors have completed studies indicating that ACRX-100 is bothsafe and efficacious in preclinical models of heart failure and CLI. Theresults of these completed and proposed preclinical studies support ourproposed Phase I/II clinical trial assessing the safety and efficacy ofACRX-100 treatment of patients with CLI.

The 66 patient combined, proposed Phase I/II study will investigate thesafety and initial efficacy of using ACRX-100 to treat Rutherford Class5 CLI patients. Safety will be monitored in each group by documentingall adverse events (AEs) and measuring standard blood laboratory values(e.g. complete blood count, plasma SDF-1 levels), with the primarysafety endpoint being the number of major AEs at 30 days (Groups 1 and2) or 60 days (Groups 3, 4 and 5) post-enrollment.

In the Phase I portion of the study (Groups 1-4), patients will receive8 or 16 injections in the upper and lower leg of either a single dosing(Groups 1 and 2) or repeat dosing sessions over 4 weeks (Groups 3 and 4)of ACRX-100 (Table 9). Dose escalation involves doubling the number ofinjections from 8 injections (Group 1) to 16 injections (Group 2),moving from a single dosing (Group 2) to 3 repeat dosing sessions (Group3), and doubling the number of injections per dosing from 8 (Group 3) to16 (Group 4). Under the assumption that repeat dosing will be moreefficacious than a single dose, Groups 3 and 4 will be randomized 2:1 toreceive either treatment or placebo (5% dextrose injection) to testpreliminary efficacy; Groups 1 and 2 will receive active drug only. Inall groups, efficacy will be evaluated over twelve months post-firstdosing by assessing the following endpoints: 1) major amputations, 2)incidence of complete wound closure, 3) survival, changes from baselinein: 4) rate of change of index ulcer healing, 5) transcutaneous oxygen(TcPO2), and 6) rest pain.

In the Phase II portion of the trial (Group 5), 30 patients will berandomized 2:1 to receive either ACRX-100 (at the more efficacious doseregimen from Group 3 or 4) or vehicle (5% dextrose solution) (Table 9).Safety and efficacy will be measured by the same endpoints as Phase Iwith the primary efficacy endpoint being major amputation free-survival.All dose escalations in Phase I and commencement of the Phase II portionof the study will only occur following DSMC review as described below.

If the Phase I/II trial results indicate that ACRX-100 is effective inimproving CLI, with the recommendation of the DSMC, patients initiallyrandomized to control groups may be offered ACRX-100 treatment at nocost to the patient under the following conditions:

1. The patient completes the trial follow-up schedule.2. The site Principal Investigator determines the patient may stillbenefit from the treatment.

If treated, the patient will be followed with a schedule consisting of(at minimum) the described safety endpoints.

TABLE 9 Proposed Phase I/II Clinical Dosing Regimen # # pDNA/ # Inj.Volume/ pDNA pDNA/ Treatment Open-label or Group Pts. Doses Dose SitesSite Conc Site times randomized 1 3 1  8 mg 8 1 ml 1 mg/ml 1 mg Day 0Open-label 2 3 1 16 mg 16 Day 0 Open-label 3 15 3  8 mg 8 Day 0, 14,Randomized 2:1 28 (10 treated, 5 control) 4 15 3 16 mg 16 Day 0, 14,Randomized 2:1 28 (10 treated, 5 control) 5 30 Best dose from Group 3and 4 Randomized 2:1 (20 treated, 10 control)

All enrolled patients will receive normal standard of care for CLI,including pharmacologic (pain medication) and wound treatment(debridement, infection control with antibiotics, etc.) as necessary.However, per inclusion/exclusion criteria, the patient will not betreated by surgical or interventional techniques, as enrolled patientsmust be poor candidates for revascularization, and must not have had arevascularization procedure within 6 weeks prior to enrollment. Onereason they are poor candidates for revascularization is that theischemia is caused by blockages in multiple arteries that cannot all bereopened or bypassed. This makes pro-angiogenic therapies such asACRX-100 attractive because they have the potential to create new bloodvessels originating from vessels throughout the ischemic leg which canrestore blood flow to a large area and thereby improve symptoms, limbfunction, and outcomes.

In the absence of therapy, these patients have a poor prognosis. CLIpatients who are poor candidates for revascularization (also termedpatients with “unreconstructable disease”) have a 40% chance ofamputation within 6 months. Furthermore, they have a 25% mortality rateat one year, including a 3-5 time increase in the risk of cardiovasculardeath compared to those without CLI.

A synopsis of the proposed Phase I/II study is provided in Table 11.Sixty-six (66) patients with non-healing ulcers (Rutherford Class 5)will be enrolled consecutively at up to 15 clinical centers. Eachpatient will receive direct intramuscular injections of ACRX-100 andfollowed for 12 months post-initial dosing. ACRX-100 will be deliveredusing a 27 gauge needle with injections spanning the thigh above theknee and the lower leg. Safety and efficacy endpoints will be collectedas outlined in Table 11. In the open label portion (Groups 1 and 2),descriptive statistics will be used to compare continuous efficacyvariables across dosing groups. Safety parameters will be collected andassessed qualitatively or summarized quantitatively by descriptivestatistics where appropriate. In the randomized portion of the study(Groups 3-5), either ACRX-100 or vehicle control will be delivered bythe same techniques as in Phase I at 8 or 16 injection sites at 0, 2 and4 weeks post-enrollment. Statistics comparing each treatment group tothe control group will be performed for all efficacy variables withstatistical significance defined as a p-value less than 0.05.

The logistics of the study are as follows. Prior to enrollment, allpatients must grant written informed consent to participate in thestudy. Patients will be screened within 2 weeks prior to planned firstinjection of ACRX-100 with testing to determine study eligibility,including ABI and Rutherford class (Table 11). Further safety testing(CMP, PT/PTT) and establishment of baseline values for efficacyendpoints (TcPO2, quality of life, ulcer size) will be performed duringthe screening period. The subject will be considered enrolled when he orshe enters the clinic in preparation for the injection procedure. In theopen-label portion (Groups 1 and 2), consecutive patients will beenrolled, and all will receive ACRX-100 treatment. In the randomizedportion (Groups 3-5), each patient will be randomized and the clinicalcenter notified of the randomization prior to the injection procedure.All patients will have follow-up at 1 week, 2 weeks, 4 weeks, 5 weeks, 3months, 6 and 12 months post-first injection to assess safety andefficacy (Table 11). For Groups 1-4, each of the first 3 patientenrollments will be separated by at least 7 days. After the final dosingof the last patient in Groups 1-4, all safety data collected during the7 days following each subject's dosing with ACRX-100 will be reviewed byan independent DSMC. The DSMC will be responsible for safety oversight,adjudication of adverse events, and review of any subject data thatmeets stopping rules. The committee will consist of a Medical Monitor(non-voting) and at least 3 other members. The DSMC must recommendescalation to the next dose in the Phase 1 portion of the study, andcommencement of the Phase II study.

Prior to submission of the CLI IND, the national Principal Investigatorand the Medical Monitor will develop a list of stopping rules which, ifany are met, will require temporarily halting trial enrollment pendingDSMC data review.

Justification of Proposed Clinical Dosing

The clinical doses proposed are based on the results of the nonclinicalsingle dose safety and efficacy study (see Example 11). As shown inTable 10 below, the proposed human starting dose is 1 mg/mL DNA perinjection site at 1 mL (8 mg total). This starting dose was based on theresults of the single dose rabbit safety and efficacy CLI study. Thestarting human dose has the same concentration and number of injectionsof an effective dose in the single dose rabbit study (1 mg/mL in 0.5 mLat 8 sites, 4 mg total). Furthermore, the starting human dose is onehalf of the maximum total DNA dose tested in the single dose rabbitstudy (4 mg/mL in 0.5 mL at 8 sites, 16 mg total). The highervolume/site used in the Phase I study, 1.0 mL, is twice the volume of0.5 mL in the nonclinical study because the human lower limb is muchlarger in muscle weight compared to the rabbit hind limb.

TABLE 10 Human and Animal Doses of ACRX-100 Highest NOAEL in EfficaciousSingle Highest Porcine Single Dose Dose Multiple Heart Human Maximum inRabbit Tested in Dose Tested Failure Starting Human Hindlimb Rabbit inRabbit Model (IND Parameter Dose Dose Ischemia Model Model # 14203) Conc(mg/mL) 1 1 1 4 1 5 Dose Vol (mL) 1 1 0.5 0.5 0.5 1 Number of Sites 8 168 8 16 20 Total DNA Dose 8 16 4 16 8 100 (mg)

The proposed CLI Phase 1 doses are also supported by the data from theporcine preclinical GLP safety and efficacy heart failure studydescribed in Example 3, above. The proposed Phase 1 CLI starting dose of8 mg total DNA provides a greater than 10-fold margin of safety relativeto the NOAEL of 100 mg found in the heart failure porcine safety study.The maximum amount of ACRX-100 proposed in the Phase 1 CLI study (16mg×3 doses) is less than half the amount of total DNA (100 mg) definedas the NOAEL for a single dose in the efficacy and safety heart failureporcine study.

Lastly, the volume per injection to be used in the Phase 1 CLI trial (1ml) is consistent with the volume used in the porcine heart failurestudy and in several published CLI clinical studies.

Following the successful completion of the Phase I/II study, either afollow-on Phase II study or a pivotal Phase III study will be designedto demonstrate the safety and efficacy of ACRX-100 at one or multipledoses in the target population of Rutherford Class 5 patients with CLI.

TABLE 11 Phase I/II Study Synopsis Protocol Title: A Phase I/II Study toEvaluate the Safety and Preliminary Efficacy of ACRX-100 Administered byDirect Intramuscular Injection to Cohorts of Adults with Critical LimbIschemia Study Phase: I/II Study Primary: To investigate the safety andtolerability of single and repeat doses of Objectives: ACRX-100delivered via direct intramuscular injections to subjects with CLISecondary: To investigate the preliminary efficacy of single and repeatdoses of ACRX-100 delivered via direct intramuscular injections tosubjects with CLI Sample Size: 66 (n = 36 Phase 1, n = 30 Phase 2) StudyMajor Inclusion Criteria: Population: Men and women 45 years of age orolder Non-healing ulcers (Rutherford category 5) with absence of woundinfection ABI of 0.4 or less Ankle systolic pressure of 70 mm Hg orless, or toe systolic pressure of 50 mm Hg or less Poor option forsurgery, angioplasty or stent placement Those diabetic subjects who areon optimal diabetes medication, with HbA1c < 8% Major ExclusionCriteria: Life expectancy of less than one year Previous majoramputation on the leg to be treated or planned major amputation withinthe first month following enrollment Evidence of osteomyelitisRevascularization with angiography evidence of improved flow in the legto be treated within 6 weeks prior to enrollment NYHA Class IV heartfailure Uncontrolled blood pressure defined as SBP ≧ 180 mmHg or DBP ≧110 mmHg despite adequate antihypertensive treatment at time ofscreening or enrollment. Known Buerger's disease Significant hepaticdisease (defined as >3-fold elevation in ALT/AST), HBV or HCV carriersActive proliferative retinopathy Immunodeficient states (e.g. known HIVpositivity, or organ transplant recipient) or subject receivingimmunosuppressive medication History of malignant neoplasm (exceptcurable non-melanoma skin malignancies) Pregnant or lactating women orpatients of childbearing potential not protected by an effective methodof birth control Presence of any other condition that, in the opinion ofthe investigator, might compromise any aspect of the trial Heartangioplasty with or without stent or CABG within 3 months prior toenrollment Major adverse cardiovascular event within 3 months prior toenrollment Previous treatment with angiogenic growth factors or withstem cell therapy within 1 year Study Design The study will be enrolledin five sequential groups with dosing and randomization characteristicsas outlined in Table 9. In Groups 1 and 2 (n = 3 each), each eligibleconsented subject will be assigned consecutively into the open enrollingcohort. Within each cohort, all patient enrollments will be separated byat least 7 days. After the final dosing of the last patient in eachcohort, all available safety data collected during the 7 days followingeach subject's treatment with ACRX-100 will be reviewed by anindependent Data Safety Monitoring Committee (DSMC). The DSMC mustrecommend escalation to the next dose. In Groups 3 and 4 (n = 15 each),patients will be randomized 2:1 to receive either ACRX- 100 or vehiclecontrol. In each group, the first three patient enrollments will beseparated by at least 7 days. After the final dosing of the last patientin each cohort, all safety data collected during the 7 days followingeach subject's treatment with ACRX-100 will be reviewed by anindependent Data Safety Monitoring Committee (DSMC). The DSMC mustrecommend escalation to the next dose (following Group 3) orcommencement of the Phase II portion of the study (following Group 4).In Group 5 (the Phase II portion of the study), 30 patients will berandomized 2:1 to receive repeat treatments of either ACRX-100 orvehicle (5% dextrose solution) at 0, 2 and 4 weeks post-enrollment.Study Methods Study methods are outlined in Table 12. Each patient willbe assessed at Day 0, 7, 14, 28, 35, 90, 180 and 360 for safety andefficacy. Study Product ACRX-100 or matching placebo Dose ACRX-100(Groups 1-5) or vehicle (Groups 3-5) will be delivered with a 27 gaugeneedle Administration and syringe at either 8 or 16 intramuscularinjections (per treatment) spanning the thigh above the knee and thelower leg. Safety and Phase 1 Primary Safety: Major adverse events at 30days post-injection Efficacy Phase 2 Primary Efficacy: Major amputationfree survival at 12 months Parameters Secondary endpoints: See Table fora complete listing. Statistical Groups 1 and 2: Descriptive parametricstatistics (mean and standard deviation) or non- Methods parametricstatistics (median and inter-quartile range) will be used to comparecontinuous efficacy variables across dosing groups. Safety parameterswill be collected and assessed qualitatively or summarizedquantitatively by descriptive statistics where appropriate. The datafrom each efficacy parameter will be assessed at each time point aseither raw values or calculated as change from baseline for eachpatient. Groups 3-5: Descriptive parametric statistics (mean andstandard deviation) or non- parametric statistics (median andinter-quartile range) will be used to compare continuous efficacyvariables between control and each dosing group. The data from eachefficacy parameter will be assessed at each time point as either rawvalues or calculated as change from baseline for each patient. A p-valueof less than 0.05 will be considered significant. Safety parameters willbe collected and assessed qualitatively or summarized quantitatively bydescriptive statistics where appropriate. Study Duration Each patientwill be followed for 12 months (360 days) after initial ACRX-100treatment. Study Centers This study will be conducted at up to 15clinical centers.

TABLE 12 Follow-up schedule for Phase I/II CLI Clinical Trial AssessmentInjection Screening Procedure Follow-up Visits Study Time Point Day −1(within 24 hours Day 0 Day Day Day 3 6 12 Day −14 of (up to 4 Day 7 1428 35 months months months to injection hours post (7 ± 1 (14 ± 1 (28 ±1 (35 ± 2 (90 ± 7 (180 ± 14 (360 ± 14 Unscheduled Day −1 procedure)dose) days) days) days) days) days) days) days) Visit Informed X ConsentInclusion/ X X Exclusion Medical X X History Concomitant X X X X X X X XX X X Meds HIV/Hep X B/C (3 ml blood drawn) Pregnancy X test (3 ml blooddrawn) Comprehensive X X Metabolic Panel (CMP) (3 ml blood drawn)Coagulation X X Tests PT/PTT and INR (3 ml blood drawn) Urinalysis X X X(post- X X X X X X X X procedure) Safety Physical X X X X X X X X X XExam Vital signs X X 2 X X X X X X X X measurements *: Pre- injection,prior to discharge Adverse X X X X X X X X X events 12 lead X X Prior toX X X X X X X X ECG discharge Plasma 4 X X X X X SDF-1 measurementsLevels (4 ml *: blood drawn) Pre- injection, 15 min, 3 hrs, Prior todischarge Complete X X Prior to X X X X X X X X Blood discharge Count (3ml blood drawn) Additional X Prior to X X X X X X X Blood discharge Drawfor Potential anti- ACRX-100 Assessment (3 ml) Injection X X X ProcedureEffectiveness Dose of X X X X X X X Pain Medications Quality of X X X XX X X life Visual X X X X X X X analog of pain intensity Ulcer X X X X XX X healing Ankle- X X X X X X X Brachial Index (ABI) Toe- X X X X X X XBrachial Index (TBI) TcPO₂ X X X X X X X Limb X X X X X X X X X SalvageSurvival X X X X X X X X X Total Blood 15 9 22 10 10 10 10 10 7 7 3Drawn (ml) (18 if female)

From the above description of the application, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

REFERENCES

-   1. Lapidot, T. and Petit, I. Exp Hematol, 2002. 30(9): p. 973-81.-   2. Askari, A. T., Unzek, S., Popovic, Z. B., Goldman, C. K., Forudi,    F., Kiedrowski, M., Rovner, A.,-   Ellis, S. G., Thomas, J. D., DiCorleto, P. E., Topol, E. J., and    Penn, M. S. Lancet, 2003. 362(9385): p. 697-703.-   3. Sasaki, T., Fukazawa, R., Ogawa, S., Kanno, S., Nitta, T., Ochi,    M., and Shimizu, K. Pediatr Int, 2007. 49(6): p. 966-71.-   4. Segers, V. F., Tokunou, T., Higgins, L. J., MacGillivray, C.,    Gannon, J., and Lee, R. T. Circulation, 2007. 116(15): p. 1683-92.-   5. Schober, A., Knarren, S., Lietz, M., Lin, E. A., and Weber, C.    Circulation, 2003. 108(20): p. 2491-7.-   6. Badillo, A. T., Chung, S., Zhang, L., Zoltick, P., and    Liechty, K. W. J Surg Res, 2007. 143(1): p. 35-42.-   7. van Weel, V., Seghers, L., de Vries, M. R., Kuiper, E. J.,    Schlingemann, R. O., Bajema, I. M., Lindeman, J. H., Delis-van    Diemen, P. M., van Hinsbergh, V. W., van Bockel, J. H., and    Quax, P. H. Arterioscler Thromb Vasc Biol, 2007. 27(6): p. 1426-32.-   8. Yano, T., Liu, Z., Donovan, J., Thomas, M. K., and Habener, J. F.    Diabetes, 2007. 56(12): p. 2946-57.-   9. Petit, I., Szyper-Kravitz, M., Nagler, A., Lahav, M., Peled, A.,    Habler, L., Ponomaryov, T., Taichman, R. S., Arenzana-Seisdedos, F.,    Fujii, N., Sandbank, J., Zipori, D., and Lapidot, T. Nat    Immunol, 2002. 3(7): p. 687-94.-   10. Gallagher, K. A., Liu, Z. J., Xiao, M., Chen, H., Goldstein, L.    J., Buerk, D. G., Nedeau, A., Thom, S. R., and Velazquez, O. C. J    Clin Invest, 2007. 117(5): p. 1249-59.-   11. Hiasa, K., Ishibashi, M., Ohtani, K., Inoue, S., Zhao, Q.,    Kitamoto, S., Sata, M., Ichiki, T., Takeshita, A., and Egashira, K.    Circulation, 2004. 109(20): p. 2454-61.-   12. Yamaguchi, J., Kusano, K. F., Masuo, 0., Kawamoto, A., Silver,    M., Murasawa, S., Bosch-Marce, M., Masuda, H., Losordo, D. W.,    Isner, J. M., and Asahara, T. Circulation, 2003. 107(9): p. 1322-8.-   13. Keo, H. H., Hirsch, A. T., Baumgartner, I., Nikol, S.,    Henry, T. D. Vascular Disease Management, 2009. 6(4): p. 118-124.-   14. Norgren, L., Hiatt, W. R., Dormandy, J. A., Nehler, M. R.,    Harris, K. A., and Fowkes, F. G. J Vasc Surg, 2007. 45 Suppl S: p.    S5-67.-   15. Dormandy, J. A. and Rutherford, R. B. J Vase Surg, 2000. 31(1 Pt    2): p. S1-S296.-   16. Selvin, E. and Erlinger, T. P. Circulation, 2004. 110(6): p.    738-43.-   17. Ostchega, Y., Paulose-Ram, R., Dillon, C. F., Gu, Q., and    Hughes, J. P. J Am Geriatr Soc, 2007. 55(4): p. 583-9.-   18. Lepantalo, M. and Matzke, S. Eur J Vasc Endovasc Surg, 1996.    11(2): p. 153-7.-   19. Ho, T. K., Tsui, J., Xu, S., Leoni, P., Abraham, D. J., and    Baker, D. M. J Vase Surg, 2010. 51(3): p. 689-699.-   20. Shah, P. B. and Losordo, D. W. Adv Genet, 2005. 54: p. 339-61.-   21. Fortuin, F. D., Vale, P., Losordo, D. W., Symes, J., DeLaria, G.    A., Tyner, J. J., Schaer, G. L., March, R., Snell, R. J., Henry, T.    D., Van Camp, J., Lopez, J. J., Richenbacher, W., Isner, J. M., and    Schatz, R. A. Am J Cardiol, 2003. 92(4): p. 436-9.-   22. Kusano, K. F., Pola, R., Murayama, T., Curry, C., Kawamoto, A.,    Iwakura, A., Shintani, S., Ii, M., Asai, J., Tkebuchava, T., Thorne,    T., Takenaka, H., Aikawa, R., Goukassian, D., von Samson, P.,    Hamada, H., Yoon, Y. S., Silver, M., Eaton, E., Ma, H., Heyd, L.,    Kearney, M., Munger, W., Porter, J. A., Kishore, R., and    Losordo, D. W. Nat Med, 2005. 11(11): p. 1197-204.-   23. Symes, J. F., Losordo, D. W., Vale, P. R., Lathi, K. G.,    Esakof, D. D., Mayskiy, M., and Isner, J. M. Ann Thorac Surg, 1999.    68(3): p. 830-6; discussion 836-7.-   24. Losordo, D. W., Vale, P. R., Hendel, R. C., Milliken, C. E.,    Fortuin, F. D., Cummings, N., Schatz, R. A., Asahara, T., Isner, J.    M., and Kuntz, R. E. Circulation, 2002. 105(17): p. 2012-8.-   25. Comerota, A. J., Throm, R. C., Miller, K. A., Henry, T.,    Chronos, N., Laird, J., Sequeira, R., Kent, C. K., Bacchetta, M.,    Goldman, C., Salenius, J. P., Schmieder, F. A., and Pilsudski, R. J    Vasc Surg, 2002. 35(5): p. 930-6.-   26. Powell, R. J., Simons, M., Mendelsohn, F. O., Daniel, G.,    Henry, T. D., Koga, M., Morishita, R., and Annex, B. H.    Circulation, 2008. 118(1): p. 58-65.-   27. Asahara, T., Chen, D., Tsurumi, Y., Kearney, M., Rossow, S.,    Passeri, J., Symes, J. F., and Isner, J. M. Circulation, 1996.    94(12): p. 3291-302.-   28. Makinen, K., Manninen, H., Hedman, M., Matsi, P., Mussalo, H.,    Alhava, E., and Yla-Herttuala, S. Mol Ther, 2002. 6(1): p. 127-33.-   29. Rajagopalan, S., Mohler, E. R., 3rd, Lederman, R. J.,    Mendelsohn, F. O., Saucedo, J. F., Goldman, C. K., Blebea, J.,    Macko, J., Kessler, P. D., Rasmussen, H. S., and Annex, B. H.    Circulation, 2003. 108(16): p. 1933-8.-   30. Nikol, S., Baumgartner, I., Van Belle, E., Diehm, C., Visona,    A., Capogrossi, M. C., Ferreira-Maldent, N., Gallino, A., Wyatt, M.    G., Wijesinghe, L. D., Fusari, M., Stephan, D., Emmerich, J.,    Pompilio, G., Vermassen, F., Pham, E., Grek, V., Coleman, M., and    Meyer, F. Mol Ther, 2008. 16(5): p. 972-8.-   31. Damas, J. K., Waehre, T., Yndestad, A., Ueland, T., Muller, F.,    Eiken, H. G., Holm, A. M., Halvorsen, B., Froland, S. S., Gullestad,    L., and Aukrust, P. Circulation, 2002. 106(1): p. 36-42.-   32. Deglurkar, I., Mal, N., Mills, W. R., Popovic, Z. B., McCarthy,    P., Blackstone, E. H., Laurita, K. R., and Penn, M. S. Hum Gene    Ther, 2006. 17(11): p. 1144-51.-   33. Zhang, G., Nakamura, Y., Wang, X., Hu, Q., Suggs, L. J., and    Zhang, J. Tissue Eng, 2007. 13(8): p. 2063-71.-   34. Zhang, M., Mal, N., Kiedrowski, M., Chacko, M., Askari, A. T.,    Popovic, Z. B., Koc, O. N., and Penn, M. S. FASEB J, 2007.    21(12): p. 3197-207.-   35. Shyu, W. C., Lin, S. Z., Yen, P. S., Su, C. Y., Chen, D. C.,    Wang, H. J., and Li, H. J Pharmacol Exp Ther, 2008. 324(2): p.    834-49.-   36. Lotan, D., Sheinberg, N., Kopolovic, J., and Dekel, B. Pediatr    Nephrol, 2008. 23(1): p. 71-7.-   37. Tang, Y. L., Qian, K., Zhang, Y. C., Shen, L., and    Phillips, M. I. Regul Pept, 2005. 125(1-3): p. 1-8.-   38. Today, M. N., Bioheart Launches First US FDA Approved Clinical    Trial That Tests Gene-Modified Stem Cell Therapy In Patients With    Congestive Heart Failure. 2010.-   39. Rajagopalan, S., Mohler, E., 3rd, Lederman, R. J., Saucedo, J.,    Mendelsohn, F. O., Olin, J., Blebea, J., Goldman, C.,    Trachtenberg, J. D., Pressler, M., Rasmussen, H., Annex, B. H., and    Hirsch, A. T. Am Heart J, 2003. 145(6): p. 1114-8.-   40. Rajagopalan, S., Shah, M., Luciano, A., Crystal, R., and    Nabel, E. G. Circulation, 2001. 104(7): p. 753-5.-   41. Schachinger, V., Erbs, S., Elsasser, A., Haberbosch, W.,    Hambrecht, R., Holschermann, H., Yu, J., Corti, R., Mathey, D. G.,    Hamm, C. W., Suselbeck, T., Werner, N., Haase, J., Neuzner, J.,    Germing, A., Mark, B., Assmus, B., Tonn, T., Dimmeler, S., and    Zeiher, A. M. Eur Heart J, 2006. 27(23): p. 2775-83.-   42. Wollert, K. C., Meyer, G. P., Lotz, J., Ringes-Lichtenberg, S.,    Lippolt, P., Breidenbach, C., Fichtner, S., Korte, T., Hornig, B.,    Messinger, D., Arseniev, L., Hertenstein, B., Ganser, A., and    Drexler, H. Lancet, 2004. 364(9429): p. 141-8.-   43. Pfeffer, M. A., McMurray, J. J., Velazquez, E. J., Rouleau, J.    L., Kober, L., Maggioni, A. P., Solomon, S. D., Swedberg, K., Van de    Werf, F., White, H., Leimberger, J. D., Henis, M., Edwards, S.,    Zelenkofske, S., Sellers, M. A., and Califf, R. M. N Engl J    Med, 2003. 349(20): p. 1893-906.-   44. Pitt, B., Remme, W., Zannad, F., Neaton, J., Martinez, F.,    Roniker, B., Bittman, R., Hurley, S., Kleiman, J., and Gatlin, M. N    Engl J Med, 2003. 348(14): p. 1309-21.-   45. Abbott, J. D., Huang, Y., Liu, D., Hickey, R., Krause, D. S.,    and Giordano, F. J. Circulation, 2004. 110(21): p. 3300-5.-   46. Elmadbouh, I., Haider, H., Jiang, S., Idris, N. M., Lu, G., and    Ashraf, M. J Mol Cell Cardiol, 2007. 42(4): p. 792-803.-   47. Rabbany, S. Y., Pastore, J., Yamamoto, M., Miller, T. J., Rafii,    S., Aras, R., and Penn, M. Cell Transplant., 2009. In press.-   48. Taniyama, Y., Tachibana, K., Hiraoka, K., Namba, T., Yamasaki,    K., Hashiya, N., Aoki, M., Ogihara, T., Yasufumi, K., and    Morishita, R. Circulation, 2002. 105(10): p. 1233-9.-   49. Flugelman, M. Y., Jaklitsch, M. T., Newman, K. D., Casscells,    W., Bratthauer, G. L., and Dichek, D. A. Circulation, 1992.    85(3): p. 1110-7.-   50. Penn, M. S. Circ Res, 2009. 104(10): p. 1133-5.-   51. Bhatt, D. L., Steg, P. G., Ohman, E. M., Hirsch, A. T., Ikeda,    Y., Mas, J. L., Goto, S., Liau, C. S., Richard, A. J., Rother, J.,    and Wilson, P. W. JAMA, 2006. 295(2): p. 180-9.-   52. Baumgartner, I., Chronos, N., Comerota, A., Henry, T.,    Pasquet, J. P., Finiels, F., Caron, A., Dedieu, J. F., Pilsudski,    R., and Delaere, P. Mol Ther, 2009. 17(5): p. 914-21.    Selected sequences disclosed:

SEQ ID NO: 1KPVSLLYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKSEQ ID NO: 2MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKSEQ ID NO: 3MDAKVVAVLALVLAALCISDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKSNNRQVCIDPKLKWIQEYLDKALNKSEQ ID NO: 4gccgcactttcactctccgtcagccgcattgcccgctcggcgtccggcccccgacccgcgctcgtccgcccgcccgcccgcccgcccgcgccatgaacgccaaggtcgtggtcgtgctggtcctcgtgctgaccgcgctctgcctcagcgacgggaagcccgtcagcctgagctacagatgcccatgccgattatcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagtaagcacaacagccaaaaaggactttccgctagacccactcgaggaaaactaaaaccttgtgagagatgaaagggcaaagacgtgggggagggggccttaaccatgaggaccaggtgtgtgtgtggggtgggcacattgatctgggatcgggcctgaggtttgccagcatttagaccctgcatttatagcatacggtatgatattgcagcttatattcatccatgccctgtacctgtgcacgttggaacttttattactggggtttttctaagaaagaaattgtattatcaacagcattttcaagcagttagttccttcatgatcatcacaatcatcatcattctcattctcattttttaaatcaacgagtacttcaagatctgaatttggcttgtttggagcatctcctctgctcccctggggagtctgggcacagtcaggtggtggcttaacagggagctggaaaaagtgtcctttcttcagacactgaggctcccgcagcagcgcccctcccaagaggaaggcctctgtggcactcagataccgactggggctgggcgccgccactgccttcacctcctctttcaacctcagtgattggctctgtgggctccatgtagaagccactattactgggactgtgctcagagacccctctcccagctattcctactctctccccgactccgagagcatgcttaatcttgcttctgcttctcatttctgtagcctgatcagcgccgcaccagccgggaagagggtgattgctggggctcgtgccctgcatccctctcctcccagggcctgccccacagctcgggccctctgtgagatccgtctttggcctcctccagaatggagctggccctctcctggggatgtgtaatggtccccctgcttacccgcaaaagacaagtctttacagaatcaaatgcaattttaaatctgagagctcgctttgagtgactgggttttgtgattgcctctgaagcctatgtatgccatggaggcactaacaaactctgaggtttccgaaatcagaagcgaaaaaatcagtgaataaaccatcatcttgccactaccccctcctgaagccacagcagggtttcaggttccaatcagaactgttggcaaggtgacatttccatgcataaatgcgatccacagaaggtcctggtggtatttgtaactttttgcaaggcatttttttatatatatttttgtgcacatttttttttacgtttctttagaaaacaaatgtatttcaaaatatatttatagtcgaacaattcatatatttgaagtggagccatatgaatgtcagtagtttatacttctctattatctcaaactactggcaatttgtaaagaaatatatatgatatataaatgtgattgcagcttttcaatgttagccacagtgtattttttcacttgtactaaaattgtatcaaatgtgacattatatgcactagcaataaaatgctaattgtttcatggtataaacgtcctactgtatgtgggaatttatttacctgaaataaaattcattagttgttagtgatggagcttaaaaaaaaSEQ ID NO: 5ccatggacgccaaggtcgtcgctgtgctggccctggtgctggccgcgctctgcatcagtgacggtaagccagtcagcctgagctacagatgcccctgccgattctttgagagccatgtcgccagagccaacgtcaaacatctgaaaatcctcaacactccaaactgtgcccttcagattgttgcaaggctgaaaagcaacaacagacaagtgtgcattgacccgaaattaaagtggatccaagagtacctggacaaagccttaaacaagtaagcacaacagcccaaaggactt

What is claimed:
 1. A method of treating critical limb ischemia in apatient comprising administering a preparation comprising a stromalcell-derived factor-1 (SDF-1) plasmid and a pharmaceutically acceptablecarrier to an affected limb of the patient, wherein said SDF-1 plasmidhas the polynucleotide sequence of the plasmid deposited with theAmerican Type Culture Collection under accession number PTA-13320. 2.The method according to claim 1, wherein said preparation isadministered to the patient by intramuscular injection directly into theaffected limb.
 3. The method according to claim 1, wherein saidpharmaceutically acceptable carrier is 5% dextrose.
 5. The methodaccording to claim 1, wherein said preparation comprises from about 0.33mg/ml to about 5 mg/ml of said SDF-1 plasmid.
 6. The method according toclaim 5, comprising about 2 mg/ml of said SDF-1 plasmid.
 7. The methodaccording to claim 5, comprising about 1 mg/ml of said SDF-1 plasmid. 8.The method according to claim 1, wherein the preparation is administeredin about 5 to about 20 injections and each injection has a volume of atleast about 1.0 ml.
 9. The method according to claim 8, wherein theamount of SDF-1 plasmid that is administered is from about 5 to about 20mg.
 10. The method according to claim 1, wherein the administration isrepeated up to three times, with the administrations each beingseparated by a period of about 2 weeks.